Ribonucleotide reductase inhibition restores platinum-sensitivity in platinum-resistant ovarian cancer: a Gynecologic Oncology Group Study

Abstract

Background: The potent ribonucleotide reductase (RNR) inhibitor 3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) was tested as a chemosensitizer for restored cisplatin-mediated cytotoxicity in platinum-resistant ovarian cancer.

Methods: Preclinical in vitro platinum-resistant ovarian cancer cell survival, RNR activity, and DNA damage assays were done after cisplatin or cisplatin plus 3-AP treatments. Six women with platinum-resistant ovarian cancer underwent four-day 3-AP (96 mg/m(2), day one to four) and cisplatin (25 mg/m(2), day two and three) infusions every 21 days until disease progression or adverse effects prohibited further therapy. Pre-therapy ovarian cancer tissues were analyzed by immunohistochemistry for RNR subunit expression as an indicator of cisplatin plus 3-AP treatment response.

Results: 3-AP preceding cisplatin exposure in platinum-resistant ovarian cancer cells was not as effective as sequencing cisplatin plus 3-AP together in cell survival assays. Platinum-mediated DNA damage (i.e., γH2AX foci) resolved quickly after cisplatin-alone or 3-AP preceding cisplatin exposure, but persisted after a cisplatin plus 3-AP sequence. On trial, 25 four-day overlapping 3-AP and cisplatin cycles were administered to six women (median 4.2 cycles per patient). 3-AP-related methemoglobinemia (range seven to 10%) occurred in two (33%) of six women, halting trial accrual.

Conclusions: When sequenced cisplatin plus 3-AP, RNR inhibition restored platinum-sensitivity in platinum-resistant ovarian cancers. 3-AP (96 mg/m(2)) infusions produced modest methemoglobinemia, the expected consequence of ribonucleotide reductase inhibitors disrupting collateral proteins containing iron.

Trial registry: ClinicalTrials.gov NCT00081276.

Figures

Figure 1

3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) lowered 6-hour deoxycytidine triphosphates (dCTP) pools and was associated with elevated 24-hour posttherapy M2 and M2b protein. Panel A: SKOV3 and OVCAR3 cells were treated with cisplatin (5 μM) and/or 3-AP (5 μM) for 6 hours and assayed for dCTP pool by a DNA polymerase extension assay. Cisplatin treatment elevated 6-hour post therapy dCTP levels, indirectly suggesting a rise in RNR activity. dCTP levels 6 hours after cisplatin treatment (and 18 hours after start of 3-AP treatment) were indistinguishable from dCTP levels in cisplatin alone treated cells (P = 1.0). When 3-AP was administered alone or co-administered with cisplatin, substantial reduction in dCTP levels were detected 6 hours post therapy (P < 0.001, star). Means (± standard error) are reported. Panel B: Immunoblots for M2 and M2b protein and corresponding dCTP level are depicted for control and treated cells (t = 0 h [before] and 24 h [after start of cisplatin]). M2 and M2b showed moderate increase after cisplatin treatment or after ribonucleotide reductase blockade by 3-AP. Corresponding 24-hour dCTP level after cisplatin, an indicator of rise in ribonucleotide reductase activity in response to cisplatin-mediated DNA damage, was higher than baseline (P < 0.01, star). Increased ribonucleotide reductase activity occurred in the 3-AP then cisplatin treatment (P < 0.01, star). In the three 3-AP treatment groups, 24-hour recovery of ribonucleotide reductase activity after 3-AP inactivation was found. Whether return to baseline or elevated recovery of activity happens because of transcriptional replacement of the M2 and/or M2b subunits or other mechanism is not discerned. Means (± standard error) are reported for RNR activity.

Figure 2

3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) protracts resolution of γH2AX foci. Panel A: Manual counts (mean ± standard error) of retained γH2AX foci at 24 hours post therapy (i.e., start of cisplatin exposure) are depicted for SKOV3 and OVCAR3 treated cells. Cisplatin damages DNA resulting in visible γH2AX foci [19]. Cisplatin plus 3-AP treated cells demonstrated an increased number of γH2AX foci, compared to cisplatin alone (P < 0.001, star) or 3-AP preceding cisplatin (P < 0.001, star) treatment, indicative of retained cisplatin-mediated DNA damage.

Figure 3

3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) effectively restored cisplatin sensitivity in “platinum-resistant” SKOV3 and OVCAR3 ovarian cancer cells. Panel A: Cells were treated with cisplatin (5 μM) and/or 3-AP (5 μM) for 6 hours and assayed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for cell mitochondrial viability at 24 hours after the start of cisplatin exposure. Compared to cisplatin alone, 3-AP alone, and 3-AP preceding cisplatin treatment, a significant cisplatin plus 3-AP interaction was found (P < 0.01, star). Panel B: 14-day clonogenic ovarian cancer cell survival was done using cisplatin (5 μM) and a wider therapeutic range of 3-AP (1, 5, or 10 μM). Here too a significant cisplatin plus 3-AP enhancement of cytotoxicity was seen (P < 0.001, star), when compared to 3-AP alone or 3-AP preceding cisplatin. Means (± standard error) are reported.

Figure 4

Pretherapy ovarian cancer ribonucleotide reductase (RNR) M1, M2, and M2b (p53R2) protein is associated with 3-aminopyridine-2-carboxyaldehyde-thiosemicarbazone (3-AP) then cisplatin treatment response. Panel A: Two pathologists blinded to treatment and outcome scored intensity of ribonucleotide reductase subunit staining. Corresponding best treatment responses are listed (S = stable, PD = progressive disease, PR = partial response, U = undetermined). Panel B: Progression-free survival (PFS) and overall survival are illustrated for six treated patients by 3-AP (96 mg/m2) given days 1 through 4 and cisplatin (25 mg/m2) given on days 2 and 3 every 21 days. A median 14-week PFS and median 28-week overall survival were observed.