Strong Synergistic Effects with APR-246 and Cisplatin in p53-Mutant Lung Cancer Cells Nina Mohell, PhD, Åsa Fransson, PhD, Jessica Alfredsson, PhD, Mikael von Euler, MD PhD, Ulf Björklund, PhD and Lars Abrahmsen, PhD Abstract #2523 Aprea AB, Solna, Sweden Presenter: Nina Mohell ([email protected]) www.aprea.com • Platinum compounds have been used as first-line treatment for many solid tumors including non small cell (NSCLC) and small cell (SCLC) lung cancer. However, patients with lung cancer often develop resistance and eventually die of chemotherapy refractory disease. • The mechanisms of resistance are multifactorial, but two important mechanisms are mutations in the tumor suppressor protein p53 and drug-induced elevation of intracellular glutathione concentration. Mutations in p53 are common in lung cancer, ranging from 33% in adenocarcinomas to 70% in SCLC (1). • APR-246 (PRIMA-1MET) is the first compound in clinical development that reactivates mutant p53. It is a prodrug that is converted to the active form methylene quinuclidinone (MQ), a Michael acceptor that binds to mutant and/or unfolded p53 restoring the correct wild type conformation and triggering apoptosis (2). • APR-246 has yielded promising results in a first-in-human clinical trial in patients with hematological malignancies and prostate cancer (3), and a Phase Ib/II study in combination with platinum-based therapy in ovarian cancer is ongoing (4). • We have previously shown strong synergy with APR-246 and platinum compounds in p53-mutant drug-resistant ovarian cancer cells (5). Moreover, APR-246 completely restored the sensitivity of cisplatin to p53-mutant resistant ovarian cancer cells (6). • The aim of the current study was to investigate whether synergistic effect with APR-246 and cisplatin can also be observed in p53-mutant lung cancer cells, and whether APR-246 can sensitize the cisplatin-resistant lung cancer cells to cisplatin. Cell lines Cancer type p53 status NC I - H17 70 NS C LC R 24 8W (h o m.) +++ SS • Synergy was also found in the SCLC cell lines NCI-H196 and NCI-H889, with R175H and C242S mutations, respectively (Table 1). NCl - H1975 NS C LC R 273H (h o m.) +++ SS NCl - H59 6 NS C LC G245C (h o m.) +++ S S* • Mixed antagonist/additive/synergistic effects were observed in the p53 null cell line HOP-62 expressing no full lenght p53. In these cells the dose-response curve of APR-246 was very steep and no reliable/consistent combination results with cisplatin could be observed. NCl - H20 87 NS C LC V1 57F (h o m.) +++ S* NCl - H2110 NS C LC R1 5 8 P (h o m.) + S P C-14 NS C LC R 24 8W (h o m.) +++ SS • Synergistic effects with APR-246 were also observed with etoposide in the PC-14/CDDP cell line. P C-14/C DDP NS C LC R 24 8W (h o m.) +++ SS NCl - H378 S C LC Y163C (h o m.) + S/S S NCl - H19 6 S C LC R175H nd S NCl - H187 S C LC S241C nd Ad d NCl - H 8 8 9 S C LC C 242 S nd S/S S NCl - H10 4 8 S C LC R 273C nd SS • Fig.2 shows that co-treatment of cisplatin with APR-246, sensitized the PC-14/CDDP cancer cells to cisplatin in a dose-dependent manner. The IC50 value of cisplatin decreased from 45 to 25 µM in the presence of 60 µM APR-246. The PC-14/CDDP cell line has been derived from the parental PC-14 cell line by chronic cisplatin exposure in vitro (9). Figure 1.Synergistic effect with APR-246 and cisplatin in lung cancer cell lines. Strong synergistic effects were observed in NSCLC cell line NCI-H1770 (1a) and in the cisplatin resistant PC-14/CDDP cell line (1b), while strong synergistic effects were observed in SCLC cell line NCI-H378 (1c). All cell lines carry p53 mutations frequently occurring in lung cancer. 1A 1B NCIH1770 Results and Discussion: PC14-CDDP (hom. R248W p53) 100 APR-246 Cisplan Combinaon Combinaon 80 0.77 40 0.54 20 0.29 60 0.50 40 Cisplan alone 20 0.17 APR-246 (µM) - - - 0.8 - 0.8 1.2 - 1.2 1.8 - 1.8 2.6 - 2.6 APR-246 (µM) - - - 8.9 - 8.9 13 - 13 Cisplan (µM) - - - - 5.9 5.9 - 8.8 8.8 - 13 13 - 20 20 Cisplan (µM) - - - - 44 44 - 67 67 NCIH378 (hom. Y163C p53) 100 APR-246 Combinaon Cell viability (%) 80 20 - 20 - 100 100 + 20 µM APR-246 80 + 30 µM APR-246 + 40 µM APR-246 + 50 µM APR-246 60 + 60 µM APR-246 + 70 µM APR-246 2. Lambert JM, Gorzov P, Veprintsev DB, Söderqvist M, Segerbäck D, Bergman J, Fersht AR, Hainaut P, Wiman KG and Bykov VJ. 2009. PRIMA-1 reactivates mutant p53 by covalent binding to the core domain. Cancer Cell. 15(5):376-88. 3. Lehmann S, Bykov VJ, Ali D, Andrén O, Cherif H, Tidefelt U, Uggla B, Yachnin J, Juliusson G, Moshfegh A, Paul C, Wiman KG and Andersson PO. 2012. Targeting p53 in vivo: a first-in-human study with p53-targeting compound APR-246 in refractory hematologic malignancies and prostate cancer. J Clin Oncol. 30(29):3633-9. 4. von Euler M, Wiman KG, Gabra H, Brenton JD, Basu B, Vergote I, Gourley C, Smith A, Alfredsson J, Mohell N and Green JA. AACR abstract #CT204, 2015. Preliminary results from PiSARRO, a phase Ib/II study of APR-246, a mutant p53 reactivating small molecule, in combination with standard chemotherapy in platinum -sensitive ovarian cancer. 5. Mohell N, Alfredsson J, Uustalu M, Fransson Å, Bykov V, Wiman KG and Björklund U. AACR abstract #3448, 2013. Strong synergistic effects with cisplatin and APR246, a novel compound reactivating mutant p53, in ovarian cancer cell lines and primary cells from patients. 6. Mohell N, Alfredsson J, Fransson Å, Bykov V, von Euler M, Wiman K and Björklund U. AACR abstract #1801, 2014. APR-246, a clinical-stage mutant p53-reactivating compound resensitizes ovarian cancer cells to platinum compounds and doxorubicin. 7. Jonsson E, Fridborg H, Nygren P and Larsson R. 1998. Synergistic interactions of combinations of topotecan with standard drugs in primary cultures of human tumor cells from patients. Eur J Clin Pharmacol. 54(7):509-14. 40 8. Gibbons D, Byers L and Kurie J. 2015. Smoking, p53 mutation, and lung cancer. Mol Cancer Res. 2014 Jan;12(1):3-13. 9. Ohmori T, Morikage T, Sugimoto Y, Fujiwara Y, Kasahara K, Nishio K, Ohta S, Sasaki Y, Takahashi, T and Saijo N. 1993. The mechanism of the difference in cellular uptake of platinum derivatives in non-small cell lung cancer cell line (PC-14) and its cisplatin-resistant subline (PC-14/CDDP). Jpn J Cancer Res. 1993 Jan;84(1):83-92. 20 60 • Planning for a Phase Ib/II study in lung cancer is ongoing. 1. http://p53.free.fr The TP53 Web Site (database). 100 0.71 0 1C • Our results suggest that combination treatment with APR-246 and platinum drugs warrants further investigations in clinical studies. References (hom. R248W p53) 0.22 0 SS = strong synergy (CI < 0.5) nd = Not determined *=n=1 PC-14/CDDP Cisplan • We observed strong synergistic effect with APR-246 and cisplatin in NSCL cancer cell lines carrying homozygous p53 hotspot mutations; NCI-H1770 (R248W) (Fig. 1a), NCI-H1975 (R273H), NCIH596 (G245C), PC-14 (R248W) and PC-14/CDDP (R248W) (Fig. 1b). All these cell lines expressed a high level of p53 (Table 1). Synergy or strong synergy was also observed in the SCLC cell line NCIH378 (Fig. 1c) carrying the frequently occurring homozygous Y163C mutation, expressed at lower levels (Table 1). SCLC = small cell lung cancer NSCLC = non-small cell lung cancer Add = additive (CI = 1.0 ± 0.2) S = synergy (CI < 0.8) • Treatment with APR-246 in combination with cisplatin resulted in synergistic or strong synergistic effect in both NSCL and SCLC cancer cells, including cell lines with “all cancer” hotspot mutations as well as cell lines carrying p53 mutations occurring more frequently in lung cancer. APR-246 Cisplan Cell viability (%) 60 hom. = homozygous + = weak p53 expression +++ = strong p53 expression Conclusions: Figure 2. APR-246 resensitizes the PC-14/CDDP lung cancer cells to cisplatin. The IC50 value of cisplatin decreased from 45 µM to 25 µM. (hom. R248Q p53) 100 Cell viability (%) • Cell viability was determined with FMCA or Cell Titer-Glo assay, p53 gene status by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Combination Index (CI) was calculated according to Additive model (7); CI = 1.0 ± 0.2 indicates additive effect, CI < 0.8 synergy and CI < 0.5 strong synergy. The results are shown as mean±SEM (n=2, if not otherwise stated). Combination APR-246 cisplatin p53 protein expression 80 Methods: Table 1. Results from combination studies with APR-246 and cisplatin in lung cancer cell lines with various p53 mutations. Cell viability (%) Introduction: • Synergistic effects were found in the NSCLC cancer cell lines NCI-2087 (hom.V157F) and NCI-H2110 (hom. R158P), expressing high and low levels of p53, respectively (Table 1). These mutations are smoking-related and occur more frequently in lung cancer than in other forms of cancer (1). Many of the mutations observed in relation to tobacco exposure are due to guanine to thymine transversion (8). 0.84 0.83 40 0 0.78 20 0.50 0 APR-246 (µM) - - - 1.8 - 1.8 2.6 - 2.6 4.0 - 4.0 5.9 - 5.9 Cisplan (µM) - - - - 20 20 - 30 30 - 44 44 - 67 67 0 20 40 Cisplan (µM) 60 80 Disclosures Mohell, Fransson, Alfredsson, von Euler, Björklund and Abrahmsen: Aprea AB