APR-246 overcomes resistance to cisplatin and doxorubicin in ovarian cancer cells

Two main causes of platinum resistance are mutation in the tumor suppressor gene TP53 and drug-induced increase in intracellular glutathione concentration. Mutations in TP53 occur in about 50% of human tumors. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53 and induces apoptosis. APR-246 is a prodrug that is converted to the active compound methylene quinuclidinone (MQ), a Michael acceptor that binds to cysteine residues in mutant p53 and restores its wild-type conformation. Here, we show that MQ also binds to cysteine in glutathione, thus decreasing intracellular free glutathione concentration. We also show that treatment with APR-246 completely restores the cisplatin and doxorubicin sensitivity to p53-mutant drug-resistant ovarian cancer cells. We propose that this unique ability of APR-246/MQ to bind to cysteines in both mutant p53 and glutathione has a key role in the resensitization as well as in the outstanding synergistic effects observed with APR-246 in combination with platinum compounds in ovarian cancer cell lines and primary cancer cells. However, MQ binding to cysteines in other targets, for example, thioredoxin reductase, may contribute as well. Strong synergy was also observed with the DNA-damaging drugs doxorubicin and gemcitabine, while additive effects were found with the taxane docetaxel. Our results provide a strong rationale for the ongoing clinical study with APR-246 in combination with platinum-based therapy in patients with p53-mutant recurrent high-grade serous (HGS) ovarian cancer. More than 96% of these patients carry TP53 mutations. Combined treatment with APR-246 and platinum or other DNA-damaging drugs could allow dramatically improved therapy of a wide range of therapy refractory p53 mutant tumors.

4 , and heated to 95°C for 5 min before separated by electrophoresis on a 4-15% TGX-gel (Bio-Rad) and transferred to PVDF membrane using a semidry trans blot system (Bio-Rad). The membranes were blocked in Tris-buffered saline containing 5% w/v non-fat dry milk and 0.1% Tween-20, followed by incubation with primary antibody overnight at 4°C, and with secondary antibody for 1 h at room temperature. Antibodies were diluted in Tris-buffered saline (TBS) containing 5% w/v non-fat dry milk and 0.1% Tween-20. The proteins were visualized by enhanced chemiluminescence system (ECL prime, VWR) and detected using a CCD camera (LAS1000 Fujifilm Tokyo Japan).

Cell Lines and Cell Culture
Supplementary  Table 1a. [2][3][4][5][6][7][8] Refers to supplementary references. ^The A2780cis cells had been reported to carry a K351N mutation, but the cell line has wt p53 according to our sequencing. ^^For the IGROV-1/CDDP cell line we found two mutations of which the R337C (het.) mutation had not been previously reported. wt = wild type het. = heterozygous fs = frame shift mutation * = stop codon NSCLC = non small cell lung cancer SCLC = small cell lung cancer # RPMI 1640 + 10 % FBS + 2 mM glutamine ¤ To retain cisplatin resistance in A2780cis, 1 µM cisplatin was added to the culture media every 3 passages. ¤¤ To retain doxorubicin resistance in A2780ADR, 0.1 µM doxorubicin was added to the culture media at least once a month. The A2780cis and A2780ADR cells were drug free for at least 48 h before they were used in FMCA assay. The A2780-CP20, NCI-H1770, NCI-H1975, NCI-H1417 and NCI-H378 cell lines were authenticated by short tandem repeat (STR) analysis (IGROV-1/CDDP was not described in ATCC or DSMZ databases for cell authentification). All other cell lines were obtained directly from cell banks and passaged for fewer than 6 months after their receipt, and reauthentication was not needed.

Cell Viability Assays
The fluorometric microculture cytotoxicity (FMCA) assay: 96-well plates with v-shaped wells (Nunc) were prepared with test substances at 10x the desired concentration. The prepared substance plates were kept at -80°C until they were used. The cell suspension was seeded out with 1.5*10 4 cells/ml (3000 cells/well) -6.0*10 4 cells/ml (12000 cells/well) in thawed substance plates and incubated for 72 h in a humidified atmosphere, at 37°C and with 5% CO2. The cell plates were washed and incubated for another 40 min with 100 µl of 1 µl fluorescein diacetate (FDA) (Sigma)/ml physiological buffer (Q2) per well. 9 The Q2 buffer was kept in room temperature and before use the amount of needed buffer WST-1 assay: Cells were seeded at 3000 cells/well, 100 µl/well, in 96-well plates with flat bottom (Costar, Corning Incorporated). Following 24 h incubation at 37°C, 5% CO2, substance containing culture medium was added, 100 µl/well, giving a total volume of 200 µl/well. The plates were incubated for 72 h at 37°C, 5% CO2. Thereafter, WST-1 reagent (Roche) was added to all wells (final dilution 1:10) and the plates were incubated for 60 minutes at 37°C, 5% CO2 before the absorbance was measured in a microplate absorbance reader (Multiskan EX photometric microplate absorbance reader, Thermo). WST-1 cell viability assay is based on the cleavage of the tetrazolium salt WST-1 (4-

Analysis of Combination Results Using Additive Model
In samples with two co-incubated substances, a predicted cell viability (decimal form, i.e., % x 0.01) was calculated according to the following formula: Predicted cell viability (decimal form) = cell viability of substance 1 (decimal form) x cell viability of substance 2 (decimal form). A "combination index" (CI) was then calculated as the measured cell viability of the sample with two co-incubated substances divided by the predicted cell viability: CI = measured cell viability / predicted cell viability. CI = 1±0.2 indicates additive effect, < 0.8 indicates synergistic and < 0.5 strong synergistic effect. CI > 1.2 indicates sub-additive or antagonistic effects.

In Vivo Xenograft Efficacy Study
When tumors had reached a volume of 90-170 mm 3 , the mice were divided into four groups of 10 mice, and randomly assigned to treatment and control groups. Tumor volumes were calculated with the formula: Tumor vol = length x width2 x 0.5. The tumor growth inhibition (% TGI) was calculated with the formula % TGI = (((Tumor volume control group Day 8 -Tumor volume control group Day 1) -(Tumor volume treatment group Day 8 -Tumor vol treatment group Day 1)) / (Tumor volume control group Day 8 -Tumor volume control group Day 1)) x 100.

Evaluation of Active Caspase-3 in Tumors
The excised tumors were fixed for 24 h in 10% buffered formalin at room temperature and embedded in paraffin. Serial sections (2-4 µm thickness) were cut and stained with haematoxylin and eosin (H&E) for histopathology. For immunohistochemistry, sections were deparaffinised in xylene, hydrated in graded ethanol and tap water and transferred to PBS. Tumor sections were subjected to heat-induced epitope retrieval in citrate buffer pH 6.0 (Vector) by a pressure cooker for 3 min at 120°C and then cooled for 30 min. Endogenous peroxidase was blocked incubating the slices with 3% hydrogen peroxide in PBS for 10 min. After blocking the tissues were washed two times with PBS containing 0.05% Tween-20 (PBST) and incubated for 30 min with 10% normal goat serum (Vector) in PBST (blocking solution). Blocking solution was substituted by primary monoclonal rabbit anti-active Caspase-3 antibody (Cell Signaling) and incubated for 1 h at room temperature. Tumors were then rinsed twice with PBST and incubated for 30 min at room temperature with the DAKO EnVision + Rabbit Polymer System. After washing with PBST, sections were incubated for 1-3 min with liquid DAB + substrate chromogen system (3,3 diaminobenzidine, Dako). They were then washed three times with tap water, counterstained for 3 minutes with Gill's hematoxylin (Bio Optica), washed with tap water, dehydrated in graded ethanol/xylene and mounted.

Glutathione Assay
The Cayman kit contains GS reductase, which generates glutathione (GSH) from glutathione disulfide GSSG. Then GSH reacts with DTNB (5,5'-dithio-bis-2-(nitrobenzoic acid), Ellman's reagent), producing a yellow coloured 5-thio-2-nitrobenzoic acid (TNB), which is measured by absorbance. The rate of formation of TNB is proportional to the GSH concentration in the sample. Since GS reductase is present in the kit, the sum of glutathione and glutathione disulfide is detected. When cell diameters were measured using a cell Coulter counter the approximate observed cell volumes were 1.56 pl for A2780 and 1.55 pl for A2780-CP20, and accordingly the total GSH (GSH + 2xGSSG) mM concentrations were ~0.64 times the displayed nmol/10 6 cells concentrations.