Abstract 3507: APR-246 reactivates mutant p53 in non-small cell lung cancer cell lines and sensitizes cells for CDDP treatment under normoxic and hypoxic conditions
Authors: Deben Christophe , Deschoolmeester Vanessa , Wouters An , Van den Bossche Jolien , Jacobs Julie , Lardon Filip , Rolfo Christian , Pauwels Patrick 
Published: August 2015

Abstract
Introduction: Tumors are often characterized by inactivating mutations in the TP53 gene. Mutations in this gene occur in about 50% of non-small cell lung cancers (NSCLC), and influence the response to common chemotherapeutics like CDDP (cisplatin). In addition, as hypoxic stress, which is often found in tumors, is able to induce p53 accumulation and p53 dependent apoptosis, the reactivation of mutant p53 might induce a strong cytotoxic effect in hypoxic tumor regions. Therefore, we treated NSCLC tumor cells with APR-246, a known activator of mutant p53, under normoxic and hypoxic conditions. In addition, we combined APR-246 with CDDP, to determine whether reactivation of mutant p53 could enhance the cytotoxic response of CDDP.
Materials & Methods: The NSCLC cell line CRL-5908, harboring a R273H p53 mutation, was used. To determine the role of mutant p53 in response to APR-246, CRL-5908 was transduced with a p53 shRNA lentiviral vector to obtain a p53 deficient sub cell line. The SRB-assay was used to determine the cytotoxic effect of both mono-and combination therapies after which possible synergism was calculated by the Chou-Talalay method, both under normoxic and hypoxic conditions (0% O2, 5% CO2, 95% N2). In addition, we determined the p53 protein level as well as the mRNA and protein levels of its main transcription targets MDM2, p21, PUMA and BAX. The induction of apoptosis and cell cycle arrest were determined by flowcytometric analysis.
Results: Our results show a clear response of CRL-5908 to APR-246, which was stronger under hypoxic conditions compared to normoxic conditions (IC50: 9.5 ± 2.5 vs. 16.3 ± 3.4). We observed that CRL-5908 was less sensitive to CDDP monotherapy than the p53 wild type cell line A549 (IC50: 15.2 ± 3.3 vs. 6.2 ± 1.6). We were able to overcome this resistance to CDDP by combining the drug with APR-246 (IC20, IC40, IC60), which resulted in CDDP IC50 values of respectively 11.5 ± 4.0; 5.0 ± 2.2 and 6.5 ± 2.0 under normoxic conditions.
Discussion: Our initial results show that APR-246 monotherapy is promising for the treatment of p53 mutant NSCLC, particularly under hypoxic conditions. In addition, APR-246 seems to sensitize cells for CDDP treatment when combined simultaneously. We will further study the underlying mechanisms, like the activation of p53 and the transcription of its main transcription target MDM2, p21, PUMA and BAX as well as apoptosis and cell cycle arrest. Moreover, we will compare simultaneous treatment of CDDP and APR-246 with sequential treatment in which CDDP is followed by APR-246 treatment, and further sort out the response of this combination therapy under hypoxic conditions, as well as the role of mutant p53.
Citation Format: Christophe Deben, Vanessa Deschoolmeester, An Wouters, Jolien Van den Bossche, Julie Jacobs, Filip Lardon, Christian Rolfo, Patrick Pauwels. APR-246 reactivates mutant p53 in non-small cell lung cancer cell lines and sensitizes cells for CDDP treatment under normoxic and hypoxic conditions. [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 3507. doi:10.1158/1538-7445.AM2015-3507