Abstract 1639: Strong synergy with APR-246 and DNA-damaging drugs in primary ovarian cancer cells

Abstract

Background: Mutations in the TP53 gene occur in at least 60% of ovarian tumors and are associated with chemoresistance and poor prognosis. APR-246 (PRIMA-1MET) is the first mutant p53-reactivating compound in clinical development and has been tested as monotherapy in hematological malignancies and prostate cancer with promising results (Lehmann et al. J Clin Oncol 30, 2012). The aim of this study was to investigate the anticancer effects of APR-246 in combination with conventional chemotherapy in cancer cells isolated from ascites fluid from ovarian cancer patients. Methods: Ascites cells were purified by Ficoll and viably frozen, and the quality and purity were confirmed by May Grünwald/Giemsa staining. For some samples, immunocytochemical stainings with anti-Ber-EP4 and anti-calretinin antibodies were used to distinguish between mesothelial and cancer cells. Cell viability was assessed with FMCA assay and Combination Index (CI) calculated using Additive model. CI < 0.8 indicates synergy and CI < 0.5 strong synergy. TP53 gene status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Results: Eight of ten samples tested were from patients with recurrent ovarian cancer previously treated with platinum drugs. Cancer cells from seven patients possessed TP53 core domain missense mutations L111Q, C135Y, P151H, Y163H, C238F, P278R and R280K, respectively; two had nonsense mutations E346* and E204*, and one was wild type. All the missense mutations have been predicted to severely affect p53 tumor suppressor function. Missense mutant p53 proteins were expressed at high levels while no p53 expression was detected in cells with wild type or nonsense mutant p53. Synergistic or strong synergistic effects with APR-246 and cisplatin were observed in all ten samples tested. Synergy was also observed with the platinum analogue carboplatin and the anthracycline doxorubicin. The IC50 values for cisplatin ranged from 3 to 40 μM and for APR-246 from 5 to 37 μM. We also tested the ability of APR-246 to sensitize the primary ovarian cancer cells carrying Y163H mutant p53, to cisplatin; the IC50-value of cisplatin decreased from 10 to 2.6 μM in the presence of 6 μM APR-246. Conclusions: We observed striking synergy with APR-246 and platinum drugs or doxorubicin. These results are consistent with our previous results in ovarian cancer cell lines showing synergy not only in p53 mutant but also in p53 null cancer cell lines. In these cells, the synergy may be related to the fact that APR-246 decreases intracellular glutathione level. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with carboplatin and doxorubicin in patients with recurrent ovarian cancer and suggest that combination treatment with APR-246 and DNA-damaging drugs could allow significantly improved treatment for ovarian cancer carrying mutant p53. Citation Format: Åsa Fransson, Daria Glaessgen, Jessica Alfredsson, Klas G. Wiman, Svetlana Bajalica Lagercrantz, Nina Mohell. Strong synergy with APR-246 and DNA-damaging drugs in primary ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1639. doi:10.1158/1538-7445.AM2015-1639

Therapeutic targeting of mutant p53 in pediatric acute lymphoblastic leukemia

“…Importantly, these TP53mut leukemias were sensitive to APR-246, likely by reactivation of high levels of dysfunctional p53 accumulated in the cells. Most importantly, in line with reports in ovarian cancer, 32,39 APR-246 clearly synergized with doxorubicin in vitro, ex vivo and in vivo, re-…”

“…28,29 In addition, induction of oxidative stress has been reported as a second activity of APR-246, deriving from glutathione depletion, thioredoxin reductase inhibition and other effects. [30][31][32][33] APR-246 demonstrated pre-clinical anti-tumor activity and synergism with DNAdamaging drugs in different cancers 32,[34][35][36][37][38][39] and showed very moderate side effect profiles in a first-in-man phase I/IIa clinical trial in patients with refractory prostate cancer, acute myeloid and chronic lymphocytic leukemia, multiple myeloma and lymphoma. 40 Accordingly, APR-246 is currently investigated in ovarian and esophageal cancer, myeloid neoplasms and melanoma in phase II clinical trials 7/35 (ClinicalTrials.gov).…”

Ovarian cancers with low CIP2A tumor expression constitute an APR-246 sensitive disease subtype

“…1A). The most apparent sensitization effect was observed with APR-246 (PRIMA-1Met/Eprenetapopt) (14)(15)(16)(17)(18). Whereas, CIP2A high cells were practically insensitive to APR-246, CIP2A low HEY cells showed > 50% reduction in cell viability (Fig.…”

Tp53-Mutated Myelodysplasia and Acute Myeloid Leukemia

“…243 A recent study suggested an additional mechanism of anti-tumor activity of APR-246 through induction of oxidative stress mediated by glutathione depletion and induction of ferroptosis. 244 Preclinical studies have shown synergistic effects of APR-246 and AZA in TP53-mutated MDS and AML cells and have supported the clinical evaluation of this drug association 241 Two phase Ib/II studies have evaluated the association of Eprenetapopt with AZA; the first trial involved the enrollment of TP53-mutated MDS (with intermediate or high-risk) and AML (oligoblastic AMLs, with 20-30% of blasts); 245 the second trial involved a similar population of patients, with the exception of the admission of AML patients with any blast percentage and the administration of the two drugs Eprenetapopt and AZA for up to one year in the eventuality of a HSCT. 246 The pooled analysis of 100 patients enrolled in these two studies showed an ORR of 69%, a CR rate of 43%, a NGS TP53 mutation negativity of 40%, a MRD negativity rate of 6% and a median OS after allo-HSCT of 16.1 months.…”

“…239 This molecule displays the unique property of restoring the DNA binding capacity of p53 mutant protein and, consequently, the growth and tumorsuppressing activities of this protein. [240][241] The restoring capacity was observed for various TP53 mutants.…”