Identification of ovarian cancer patient subpopulations with increased sensitivity to targeted therapies could offer significant clinical benefit. We report that 22% of the high-grade ovarian cancer tumors at diagnosis express CIP2A oncoprotein at low levels. Furthermore, regardless of their significantly lower likelihood of disease relapse after standard chemotherapy, a portion of relapsed tumors retain their CIP2A-deficient phenotype. Through a screen for therapeutics that would preferentially kill CIP2A-deficient ovarian cancer cells, we identified reactive oxygen species inducer APR-246, tested previously in ovarian cancer clinical trials. Consistent with CIP2A-deficient ovarian cancer subtype in humans, CIP2A is dispensable for development of MISIIR-Tag–driven mouse ovarian cancer tumors. Nevertheless, CIP2A-null ovarian cancer tumor cells from MISIIR-Tag mice displayed APR-246 hypersensitivity both in vitro and in vivo. Mechanistically, the lack of CIP2A expression hypersensitizes the ovarian cancer cells to APR-246 by inhibition of NF-κB activity. Accordingly, combination of APR-246 and NF-κB inhibitor compounds strongly synergized in killing of CIP2A-positive ovarian cancer cells. Collectively, the results warrant consideration of clinical testing of APR-246 for CIP2A-deficient ovarian cancer tumor subtype patients. Results also reveal CIP2A as a candidate APR-246 combination therapy target for ovarian cancer.

Ovarian cancer is the fifth most common cause of cancer-related death among females in the United States, where every year more than 22, 000 women are diagnosed with ovarian cancer, and around 14, 000 die from this disease. Although most patients with primary ovarian cancer respond well to standard adjuvant chemotherapy, the disease becomes resistant to most current therapies during progression, and the 5-year disease-specific overall survival has been historically less than 50% (1). However, as evidenced by a significant clinical benefit of PARP inhibitors for patients with platinum-sensitive ovarian cancer, identification of new therapies for patient subpopulations with enhanced therapeutic response, might significantly change the disease outcome of those patients with ovarian cancer (2).

Tumor suppressor protein phosphatase 2A (PP2A) complexes control the activities of a number of oncogenic proteins and cancer driver pathways (3). In many cancer types, the tumor suppressor activity of PP2A is suppressed by its endogenous inhibitor protein CIP2A (4, 5). CIP2A has a restricted expression profile in most normal human and mouse tissues (5, 6), but it is frequently overexpressed in human malignancies (4, 7). High CIP2A expression has been observed in 68% to 83% of high-grade serous ovarian cancer tumors, and this associates with high proliferation index, aneuploidy, advanced tumor grade, TP53 mutation, and EGFR expression (8, 9). The remaining 17% to 32% of patients with ovarian cancer with CIP2A-deficient tumors have significantly longer overall cancer-specific survival both in an unselected patient population, as well as among patients treated with standard platinum-based chemotherapy (8). CIP2A was recently also shown in cell culture to protect ovarian cancer cells from cisplatin-induced apoptosis (10) and to associate with stemness features in patient-derived high-grade serous cancer (HGSC) cells (11). Furthermore, in two cancer drug response screens, CIP2A depletion was shown to increase the therapeutic response of HeLa and KRAS-mutant lung cancer cells to various cancer therapies (12, 13). Together, these results indicate that CIP2A-deficient ovarian cancer tumors, constituting approximately one of five of all patients with ovarian cancer, may represent a less aggressive, and more therapy-sensitive ovarian cancer subtype. The aim of this study was to identify clinically applicable compounds that preferentially kill CIP2A-deficient ovarian cancer cells. Discovery of such compounds could potentially provide the basis for a predictive patient stratification strategy for those with ovarian cancer with CIP2A-deficient tumor subtype (2).

Patient material

The original patient cohort consisted of 562 patients treated for serous ovarian carcinoma at the Department of Obstetrics and Gynecology of the Helsinki University Central Hospital between 1964 and 2000. The study material is retrospective in nature and as a significant proportion of patients had died from their disease before initiation of the study, instead of written informed consent, the permission to use the tissue samples was obtained from the National Supervisory Authority of Welfare (No. 1251/06.01.03.01/2014). The study was approved by the Ethics Committee of the Department of Obstetrics and Gynecology (No. 040/95, No. 56/13/03/03/2014 §100) and was conducted according to the principles of Declaration of Helsinki. Consecutive patients treated for serous ovarian carcinoma were searched according to pathologic records. The serous histology of all carcinomas had originally been determined and later verified by a gynecologic pathologist. The clinical information of the patients was extracted from the hospital records, and additional survival information was obtained from the Population Register Centre of Finland. To be included in the study, data of the primary treatment and the survival status of the patient were required. Staging and grading of the tumors was carried out according to FIGO classifications.

Short hairpin RNAs and stable cell lines

Stable HEY (CLU302, Cellutions Biosystems Inc.) and TYK-NU (JCRB0234.0, JCRB) and TYK-NU.CPR (JCRB0234.1, JCRB) cell lines were generated using pGIPZ lentiviral vectors expressing GFP and puromycin resistance (Open Biosystems). Cells transfected with lentiviral vector pGIPZ.NS shRNA containing nonsilencing shRNA served as control cells expressing high CIP2A. Two stable cell lines for low CIP2A expression were generated using pGIPZ.shRNA1 (No. 556) and pGIPZ.shRNA2 (No. 557) containing targeting CIP2A antisense sequences TACATCAGCAGCAAGTTTG and TACTCAATGTCTTTATGTG, respectively. At the time of transduction, cell line confluency was approximately 40% to 50%. HEY cells were transduced with polybrene (8 μg/mL), TYK-NU cells with the combination of Fugene HD (Promega) and polybrene for 4 hours. After infection, the cells were selected for puromycin resistance, and if the number of GFP-positive cells was low, the cells were GFP sorted with FACS for further experiments. Finally, all cell lines were tested negative for replication-competent viruses (RCV test) as well as for Mycoplasma, Acholeplasma, Entomoplasma, and Spiroplasma (MycoAlert Mycoplasma Detection kit, Lonza).

Cell culture

Ovarian cancer cell lines were cultured in the appropriate cell culture media supplemented with 10% heat-inactivated FCS, 2 mmol/L l-glutamine, penicillin (50 U/mL), and streptomycin (50 μg/mL). For HEY (CLU302, Cellutions Biosystems Inc.), OVCAR-8 (OVCAR-8, NCI-DTP), and OVCAR-3 (HTB-161, ATCC) cell lines, the recommended media were RPMI1640, and for CAOV-3 (HTB-75, ATCC), SKOV-3 (HTB-77, ATCC), and TYK-NU (JCRB0234.0, JCRB) cell lines DMEM was used. All cell lines were bought from commercial sources or cell line banks as listed above and tested. All cell lines were authenticated by short tandem repeat profiling by their respective commercial source or cell line bank. On procurement of these cell lines, back-ups were tested for Mycoplasma (MycoAlert Mycoplasma Detection Kit, Lonza) and frozen in liquid nitrogen at early passages. For experiments, all cell lines were cultured for less than 2 months or 20 passages. The cells were routinely tested for negative Mycoplasma contamination according to the manufacturer's protocol (The MycoAlert Mycoplasma Detection kit, Lonza). Primary ovarian cancer cells from mouse tumors were cultured as previously described (14). Patient-derived ovarian cancer cells were cultured adherent as described in ref. 11.

APR-246 and cell-based assays

APR-246 (PRIMA-1Met/Eprenetapopt; see ref. 15 for the chemical structure) was obtained either from Tocris (catalog No. 3710; for the experiments shown as Fig. 1A and B) or as a generous gift from APREA Therapeutics (rest of the experiments). Cell viability and apoptosis were determined using CellTiter-Glo Luminescent Cell Viability Assay (Promega G7571) and Caspase-Glo 3/7 Assay (Promega G8091), respectively, according to the manufacturer's instructions. Cells were seeded on 96-well plates, incubated at 37°C/5%CO2 for 72 hours, and treated with chemotherapy drugs at chosen concentrations for 48 hours. Luminescence was read using a Wallac-1251 Luminometer. Cellular DNA damage was measured using single-cell gel electrophoresis (APR-246 treatment for 6 hours). For colony growth assays, cells were plated on 6-well plates and incubated at 37°C/5%CO2. After drug treatments, the cells were washed with PBS, fixed with ethanol, and stained with 0.5% crystal violet (Sigma C-3886) for 1 hour at room temperature, washed and scanned.

Figure 1.

Identification of APR-246 hypersensitivity in CIP2A shRNA ovarian cancer cells. A, Relative cell viability of HEY cells stably transduced either by control shRNA (HEY-CTLshRNA) or by CIP2A targeted shRNA (HEY-CIP2AshRNA) treated with indicated cancer therapeutics for 24 hours. B, Relative caspase 3/7 activity in HEY-CTLshRNA or HEY-CIP2AshRNA cells treated with indicated cancer therapeutics for 48 hours. Shown is mean ± SD of parallel samples from representative screen. C, Relative cell viability of TYK-NU cells stably transduced either by control shRNA (TYK-NU-CTLshRNA) or by two CIP2A-targeted shRNAs (TYK-NU-CIP2AshRNA1, 2) treated with increasing concentrations of APR-246 for 48 hours. Shown is mean ± SD of parallel samples from representative screen (n = 12). *, P ≤ 0.05, one-sample t and Wilcoxon test. EC50: half maximal effective concentration. D, Colony growth assay of TYK-NU-CTLshRNA and TYK-NU-CIP2AshRNA1, 2 cells and their cisplatin-resistant derivatives (TYK-NU.CPR) treated with indicated doses of APR-246. E, Relative cell viability of HEY-CTLshRNA or HEY-CIP2AshRNA cells treated with either APR-246 (20 μmol/L) alone or in combination with N-acetyl cysteine (NAC) (5 mmol/L) for 48 hours. Shown is mean ± SD of parallel samples from three independent experiments (n = 4 in each). ***, P ≤ 0.001, two-way ANOVA.

Figure 1.

Identification of APR-246 hypersensitivity in CIP2A shRNA ovarian cancer cells. A, Relative cell viability of HEY cells stably transduced either by control shRNA (HEY-CTLshRNA) or by CIP2A targeted shRNA (HEY-CIP2AshRNA) treated with indicated cancer therapeutics for 24 hours. B, Relative caspase 3/7 activity in HEY-CTLshRNA or HEY-CIP2AshRNA cells treated with indicated cancer therapeutics for 48 hours. Shown is mean ± SD of parallel samples from representative screen. C, Relative cell viability of TYK-NU cells stably transduced either by control shRNA (TYK-NU-CTLshRNA) or by two CIP2A-targeted shRNAs (TYK-NU-CIP2AshRNA1, 2) treated with increasing concentrations of APR-246 for 48 hours. Shown is mean ± SD of parallel samples from representative screen (n = 12). *, P ≤ 0.05, one-sample t and Wilcoxon test. EC50: half maximal effective concentration. D, Colony growth assay of TYK-NU-CTLshRNA and TYK-NU-CIP2AshRNA1, 2 cells and their cisplatin-resistant derivatives (TYK-NU.CPR) treated with indicated doses of APR-246. E, Relative cell viability of HEY-CTLshRNA or HEY-CIP2AshRNA cells treated with either APR-246 (20 μmol/L) alone or in combination with N-acetyl cysteine (NAC) (5 mmol/L) for 48 hours. Shown is mean ± SD of parallel samples from three independent experiments (n = 4 in each). ***, P ≤ 0.001, two-way ANOVA.

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Genetic mouse models

All animal work protocols were approved by the Project Authorisation Board of the Regional State Administrative Agency for Southern Finland. TgMISIIR-TAg transgenic mice (14) were crossed with a CIP2A genetrap mouse model (6). The resulting pups were genotyped by CIP2A PCR analysis, and expression was confirmed with RT-PCR, as described in ref. 16. Only female mice were used for the experiments. The ovarian tumor development was assessed from culled mice as described in ref. 17. Measurement of metabolic active tumor volumes (MATV; ref. 18) was used to assess APR-246 treatment response from PET images. Twenty-minute–long static scans were performed in fasted (4 hours) mice at 2 hours postinjection of fluorine-18–labeled fluorodeoxyglucose (18F-FDG, 5 MBq, i.v.) twice, prior treatment and 2 days after the last injection. Three-dimensional imaging data were then reconstructed with 2D filtered back projection, resulting in images with a final voxel size of 0.78 × 0.78 × 0.80 mm. 18F-FDG uptake was measured as percentage of injected dose per gram tissue (% ID/g). Volumes of interest were defined (Inveon Research Workplace analysis software, Siemens Medical Solutions) to obtain the MATV using a fixed % ID/g value of 2.5 as a threshold value, which covered the entire tumor in all cases. To assess potential in vivo toxicity of APR-246, both wild-type (WT) and CIP2A-deficient mice were treated with APR-246 for 7 days after which the indicated tissues were evaluated by visual inspection and weighting of the tissue.

Data availability

RNA-sequencing data can be publicly accessed at Gene Expression Omnibus (GEO accession code: GSE195984). Other original data will be available by request.

Additional methods are described in the Supplementary Materials.

Screening for therapeutics that preferentially kill CIP2A-deficient ovarian cancer cells

In a previously described retrospective cohort of 562 patients with serous ovarian cancer treated with standard chemotherapy (8), and for which both the CIP2A status by IHC, and relapse status was known, 266 patients achieved complete response (CR) after surgery and six to eight rounds of paclitaxel–carboplatin combination. Among this group, 21.4% of tumors had negative CIP2A protein expression (Supplementary Table S1). Notably, patients with a CIP2A-negative ovarian cancer tumor at diagnosis significantly more often achieved complete response (CR) than patients with a CIP2A-positive tumor (57% vs. 45%, χ2 test, P = 0.044). CIP2A negativity also very significantly predicted lower likelihood for disease relapse after chemotherapy (Supplementary Table S1).

These results indicate that a portion of ovarian cancer tumors develop in a CIP2A-independent manner. The results also support the earlier findings that CIP2A-deficient tumors could constitute a more therapy-sensitive subtype (10, 12, 13). To identify potential novel therapies for the CIP2A-deficient ovarian cancer subtype, we conducted a drug screen comparing cell viability effects of both clinically used and experimental drugs between HEY-CTLshRNA (control shRNA) and HEY-CIP2AshRNA (CIP2A shRNA) cells. Inhibition of CIP2A expression was confirmed by Western blotting (Supplementary Fig. S1A). HEY-CTLshRNA cells showed multidrug resistance against chemotherapies commonly used for ovarian cancer (cisplatin, doxorubicin, olaparib, paclitaxel, topotecan; Fig. 1A). However, HEY-CIP2AshRNA cells were at least to a certain extent more sensitive to the majority of the tested drugs at chosen concentrations (Fig. 1A). The most apparent sensitization effect was observed with APR-246 (PRIMA-1Met/Eprenetapopt; refs. 15, 19–22). Whereas HEY-CTLshRNA cells were practically insensitive to APR-246, HEY-CIP2AshRNA cells showed a >50% reduction in cell viability (Fig. 1A). APR-246 (Eprenetapopt) has been studied in a clinical ovarian cancer trial (23), and it showed promising clinical activity in a recent phase II trial in acute myeloid leukemia (AML; ref. 22).

To validate these results, and to understand the mode of cell killing by APR-246 in HEY-CIP2AshRNA cells, we screened nine of the drugs with a caspase 3/7 apoptosis assay. The HEY cells were resistant to 17-AAG, cisplatin, paclitaxel, and dasatinib, regardless of their CIP2A status (Fig. 1B). On the other hand, docetaxel, doxorubicin, gemcitabine, and UCN-01 induced caspase 3/7 activity in HEY-CTLshRNA cells. Notably, APR-246 was the only drug that did not induce apoptosis in HEY-CTLshRNA cells but showed clearly higher apoptotic response in HEY-CIP2A shRNA cells (Fig. 1B). Apoptosis induction in APR-246–treated HEY-CIP2AshRNA cells was confirmed by the COMET assay by using two independent shRNA sequences (Supplementary Fig. S1B and S1C).

To confirm that vulnerability of CIP2A-silenced cells to APR-246 was not restricted to HEY cells, we tested the impact of CIP2A for APR-246 response in the HGSC cell line TYK-NU. In a cell viability assay, TYK-NU-CIP2AshRNA cells showed dramatically decreased EC50 values for APR-246 as compared with control shRNA–expressing cells, and there was no difference between CIP2A-silenced cells expressing two independent CIP2A shRNA sequences (Fig. 1C). Hypersensitivity of CIP2A-silenced cells to APR-246 was also confirmed by colony growth assays in TYK-NU, and its cisplatin-resistant derivative TYK-NU.CPR cell line (Fig. 1D). To confirm that the effects were not related to clonal selection of shRNA-transduced cells and to expand the results to yet other ovarian cancer cell lines, we transiently inhibited CIP2A expression by siRNA transfection in HEY, CAOV-3, NIH:OVCAR3, SKOV-3, and OVCAR-8 cells. In all cell lines, transient CIP2A silencing resulted in increased sensitivity to APR-246 in a cell viability assay (Supplementary Fig. S1D).

Although APR-246 was originally identified as a compound that reactivates mutant TP53 (15, 20, 24), the tested ovarian cancer cell lines displaying hypersensitivity to APR-246 upon CIP2A inhibition, exhibit varying TP53 mutation status. HEY is TP53 WT, and SKOV-3 has both TP53 alleles deleted, the rest of the cells lines harbor distinct TP53 mutations: TYK-NU (R175H); NIH:OVCAR3 (R248Q); CAOV-3 (Q136*); and OVCAR8 (Y126_K132del; c.376–396del) (ref. 25; https://p53.iarc.fr; https://web.expasy.org/cellosaurus/). Therefore, it is unlikely that the cell killing effects by APR-246 in the tested ovarian cancer cells would be mediated solely by its mutant TP53 reactivating activity. On the other hand, several recent studies (using some of the same ovarian cancer cells as mentioned here) have shown that APR-246 kills cancer cells independently of TP53 but via induction of reactive oxygen species (ROS; refs. 20, 25, 26). Moreover, a recent study showed that MQ, the active product of APR-246 in cells, conjugates with GSH to disrupt the cellular antioxidant balance (21). In a similar vein, we observed APR-246–elicited induction of ROS production in HEY cells, and this was completely quenched by pretreatment of cells with antioxidant N-acetyl cysteine (NAC; Supplementary Fig. S2A). We also validated ROS induction in both control and CIP2A-silenced cells upon APR-246 treatment. Although slightly higher basal ROS levels were detected in CIP2A-silenced clones, a clear APR-246–elicited induction of ROS was detected in all cell lines (Supplementary Fig. S2B). Strongly supporting ROS induction as a causative mechanism for APR-246–elicited killing of CIP2A-silenced cells, NAC pretreatment prevented the effects of APR-246 in cell viability (Fig. 1E). Of a note, the high micromolar concentrations of APR-246 required for ovarian cancer cell killing is consistent with published studies (21, 25) and due to intracellular metabolism of the drug to the active product methylene quinuclidinone (MQ; ref. 21). Furthermore, experiments shown in Fig. 1A and B were performed with drug patch that apparently had lower bioactivity, and hence up to 100 μmol/L concentrations had to be used, whereas the rest of the experiments were performed with APR-246 provided generously by APREA Therapeutics developing APR-246 (Eprenetapopt) toward clinical cancer therapy.

Collectively, these results identify low CIP2A expression as a vulnerability to APR-246 across multiple chemotherapy-resistant ovarian cancer cell lines.

Low CIP2A expression confers ovarian cancer cell APR-246 hypersensitivity in vivo

In vivo relevance of CIP2A on ovarian cancer cell APR-246 sensitivity was assessed by a subcutaneous xenograft assay with stable shRNA–transduced HEY cells. The tumor growth of HEY cells was not affected by CIP2A shRNA, or if anything, the HEY-CIP2AshRNA xenografts grew slightly larger during the follow-up period (Fig. 2A, B). Consistent with resistance of CTLshRNA cells to APR-246 in vitro (Fig. 1), tumor growth of HEY-CTLshRNA cells in vivo was indistinguishable between vehicle (PBS) and APR-246–treated mice (Fig. 2A). Instead, APR-246 therapy significantly decreased tumor growth of HEY-CIP2AshRNA cells (Fig. 2B). Notably, while HEY-CIP2AshRNA cells were confirmed to have almost negligible CIP2A protein expression upon transplantation (Fig. 2C), the xenograft tumors from control, or HEY-CIP2AshRNA cells were indistinguishable for their CIP2A IHC positivity at the end of the in vivo therapy experiment (Fig. 2D). These results indicate that the CIP2A positivity in the rare population of HEY-CIP2AshRNA cells provided a strong selection advantage against APR-246 therapy.

Figure 2.

Low CIP2A expression confers ovarian cancer cell APR-246 hypersensitivity in vivo. A and B, Antitumor efficacy of APR-246 in HEY-CTLshRNA or HEY-CIP2AshRNA cell xenografts. Cells were injected subcutaneously in the immunocompromised mice and APR-246 treatment (5 days per week) was started after the average tumor size reached 100 mm3. Shown is average tumor size from five mice in the group ± SD. *, P ≤ 0.05, t test. C, Western blot analysis of CIP2A expression levels from HEY-CTLshRNA or HEY-CIP2AshRNA cells before inoculation as xenografts. D, CIP2A IHC analyses of representative endpoint APR-246–treated xenograft tumors from A and B. E, Representative ovarian tumors from mice with indicated genotypes. F and G, APR-246 ex vivo sensitivity of primary TgMISIIR-Tag murOVCAR cell lines with indicated CIP2A genotypes (n = 6). In statistical analysis of F, HOZ cells were compared with WT and HEZ cells at all APR-246 concentrations. *, P ≤ 0.05, one-sample t and Wilcoxon test. H, Pretreatment of TgMISIIR-Tag murOVCAR cells with ROS scavenger NAC rescues CIP2A(HOZ) murOVCAR cells from APR-246–induced cell death. Ten μmol/L APR-246. I, PET/CT images of mice bearing TgMISIIR-Tag X CIP2A WT (top) and TgMISIIR-Tag X HOZ (bottom) tumors before and after treatment with APR-246 (100 mg/kg for 2 weeks (5 days per week)). Twenty-minute-long scans were performed 120 minutes postinjection of 5 MBq [18F]FDG (i.v). Tumors are highlighted with red circles. J, Percent change in metabolic active tumor volumes (MATVs) between mice scanned before APR-246 treatment and two days after the last drug injection. K, Average percent change in tumor volume in response to APR-246 therapy in mice with indicated genotypes (n = 5–6). *, P ≤ 0.05, t test.

Figure 2.

Low CIP2A expression confers ovarian cancer cell APR-246 hypersensitivity in vivo. A and B, Antitumor efficacy of APR-246 in HEY-CTLshRNA or HEY-CIP2AshRNA cell xenografts. Cells were injected subcutaneously in the immunocompromised mice and APR-246 treatment (5 days per week) was started after the average tumor size reached 100 mm3. Shown is average tumor size from five mice in the group ± SD. *, P ≤ 0.05, t test. C, Western blot analysis of CIP2A expression levels from HEY-CTLshRNA or HEY-CIP2AshRNA cells before inoculation as xenografts. D, CIP2A IHC analyses of representative endpoint APR-246–treated xenograft tumors from A and B. E, Representative ovarian tumors from mice with indicated genotypes. F and G, APR-246 ex vivo sensitivity of primary TgMISIIR-Tag murOVCAR cell lines with indicated CIP2A genotypes (n = 6). In statistical analysis of F, HOZ cells were compared with WT and HEZ cells at all APR-246 concentrations. *, P ≤ 0.05, one-sample t and Wilcoxon test. H, Pretreatment of TgMISIIR-Tag murOVCAR cells with ROS scavenger NAC rescues CIP2A(HOZ) murOVCAR cells from APR-246–induced cell death. Ten μmol/L APR-246. I, PET/CT images of mice bearing TgMISIIR-Tag X CIP2A WT (top) and TgMISIIR-Tag X HOZ (bottom) tumors before and after treatment with APR-246 (100 mg/kg for 2 weeks (5 days per week)). Twenty-minute-long scans were performed 120 minutes postinjection of 5 MBq [18F]FDG (i.v). Tumors are highlighted with red circles. J, Percent change in metabolic active tumor volumes (MATVs) between mice scanned before APR-246 treatment and two days after the last drug injection. K, Average percent change in tumor volume in response to APR-246 therapy in mice with indicated genotypes (n = 5–6). *, P ≤ 0.05, t test.

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To further assess the in vivo relevance of CIP2A for APR-246 therapy response, the heterozygous and homozygous CIP2A-deficient mice (CIP2AHEZ and CIP2AHOZ, respectively; ref. 6) were crossed to the MISIIR-TAg ovarian cancer mouse model (14). Consistent with human data that ovarian cancer tumors may develop in a CIP2A-independent manner (Supplementary Table S1), we reported recently that there is no difference in ovarian cancer tumorigenesis between MISIIR-TAg X CIP2AWT and MISIIR-TAg X CIP2AHOZ mice (ref. 17; Fig. 2E). This is also consistent with lack of inhibitory effects of CIP2A shRNA on HEY xenograft growth (Fig. 2A and B). To address whether the CIP2A-deficient tumor cells from MISIIR-TAg mouse crosses yet exhibit APR-246 hypersensitivity, the ovarian cancer cells from all three genotypes were isolated and cultured to retain their malignant characteristics as described previously (14). Fully consistent with human cell results, cells from MISIIR-TAg X CIP2AHOZ mice showed a dramatic hypersensitivity to APR-246 both in cell viability and colony growth assays (Fig. 2F, G). Also, similar to human cells, APR-246–elicited cell killing of MISIIR-TAg X CIP2AHOZ cells was fully rescued by NAC pretreatment (Fig. 2H).

Encouraged by these findings, we compared the in vivo APR-246 response of MISIIR-TAg ovarian cancer tumors in both CIP2A genotypes by metabolic active tumor volume (MATV) measurement using PET/CT-imaging (Fig. 2I). After quantification, all but one MISIIR-TAg X CIP2AHOZ tumor showed hypersensitivity to APR-246 therapy, as compared with tumors from MISIIR-TAg X CIP2AWT mice (Fig. 2J). The average percent change in tumor volume was significantly different between the genotypes (Fig. 2K). Finally, we did not observe any apparent genotype-specific differences in the weight of the mice or organs from the APR-246–treated mice, indicating that CIP2A deficiency does not result in critically limiting APR-246 hypersensitivity in the normal cells (Supplementary Fig. S3A–S3D).

These results show that ovarian cancer tumors with low CIP2A expression are hypersensitive to APR-246 therapy in vivo. However, as all the existing data related to CIP2A status in human ovarian cancer is from diagnostic samples (8–10), it is unclear whether tumors with low CIP2A expression exist among the relapsed cases. Thereby, we surveyed CIP2A protein expression from a limited number (n = 10) of available samples from HGSC ovarian cancer ascites at disease relapse. Quantification of CIP2A protein levels demonstrated that there were clear differences between samples in CIP2A protein expression (Supplementary Fig. S3E). Importantly, four of 10 of the relapsed HGSC samples (No. 5, No. 6, No. 8, and No. 10) could be clearly defined to have low CIP2A expression as compared to the rest of the tumors (Supplementary Fig. S3E). These results indicate that diagnostic identification of CIP2A status in recurrent ovarian cancer tumors could have predictive potential for these patients regarding clinical responsiveness to APR-246 currently in clinical development (22, 23).

Transcriptional profiling of APR-246 hypersensitive CIP2A silenced ovarian cancer cells

To understand the mechanistic basis of APR-246 hypersensitivity in CIP2A-silenced ovarian cancer cells, we conducted RNA-sequencing analysis between HEY-CTLshRNA and HEY-CIP2AshRNA cells. The parental HEY cells were included in the HEY-CTLshRNA cohort to increase the statistical power of the gene set enrichment analysis (GSEA) and to minimize the risk that some transcriptional changes would be solely due to viral shRNA transduction. We identified 147 genes that were underexpressed and 249 genes that were overexpressed, in HEY-CTLshRNA as compared with HEY-CIP2AshRNA cells (Fig. 3A, Log2 FC >1, P ≤ 0.05; Supplementary Table S2). In the GSEA analysis, three transcriptional programs; epithelial–mesenchymal transition (EMT), TNFA signaling via NF-κB (NF-κB), and MYC targets, were significantly associated with differential gene expression profiles between HEY-CTLshRNA and HEY-CIP2AshRNA cells (Fig. 3B). The top ranking differentially expressed genes in these transcriptional programs are displayed in Fig. 3C. Importantly, all these gene expression programs are intimately linked to ovarian cancer pathogenesis, and MYC regulation is a hallmark for CIP2A activity in cancer cells (5). On the other hand, the identified role of CIP2A in supporting NF-κB activity in ovarian cancer cells is consistent with recent results from breast cancer cells (27). Expression profiling data can be accessed at GEO (GEO accession code: GSE195984).

Figure 3.

CIP2A-dependent gene expression profiles in HEY cells. A, Volcano blot analysis of differentially expressed genes between HEY-CTLshRNA and HEY-CIP2AshRNA cells. Each dot represents one gene. Green and red dots represent significantly (Log2 ≤1 or <1; P ≤ 0.05) repressed and increased genes, respectively, in HEY-CTLshRNA versus HEY-CIP2AshRNA cells. B, GSEA analysis of differentially expressed genes between HEY-CTLshRNA (includes both parental and control shRNA cells) and HEY-CIP2AshRNA cells. C, Heatmap presentation of the top ranking differentially expressed genes from the GSEA profiles shown in B.

Figure 3.

CIP2A-dependent gene expression profiles in HEY cells. A, Volcano blot analysis of differentially expressed genes between HEY-CTLshRNA and HEY-CIP2AshRNA cells. Each dot represents one gene. Green and red dots represent significantly (Log2 ≤1 or <1; P ≤ 0.05) repressed and increased genes, respectively, in HEY-CTLshRNA versus HEY-CIP2AshRNA cells. B, GSEA analysis of differentially expressed genes between HEY-CTLshRNA (includes both parental and control shRNA cells) and HEY-CIP2AshRNA cells. C, Heatmap presentation of the top ranking differentially expressed genes from the GSEA profiles shown in B.

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CIP2A targets NF-κB to confer APR-246 resistance

Albeit changes in EMT and MYC can both contribute to drug resistance, we focused our functional validation experiments to NF-κB signaling. This was due to direct links of NF-κB to apoptosis resistance in ovarian cancer (28), and previous data that inhibition of NF-κB inhibits cellular glutathione levels thereby potentially sensitizing cells to ROS-inducing drugs such as APR-246 (29). Furthermore, the target of CIP2A, PP2A, is a tumor suppressor that inactivates NF-κB signaling via dephosphorylation (30–32). First, we validated CIP2A-elicited regulation of selected NF-κB target genes by qPCR (Supplementary Fig. S4A and S4B). Furthermore, HEY-CIP2AshRNA cells displayed significantly lower NF-κB–driven gene promoter activity (Fig. 4A). To directly assess CIP2A-mediated regulation of NF-κB, we analyzed nuclear translocation of the phosphoregulated component of the NF-κB complex, p65, between HEY-CTLshRNA and HEY-CIP2AshRNA cells. HEY-CTLshRNA cells had a significantly higher proportion of nuclear p65 than HEY-CIP2AshRNA cells in both control and TNFα-treated cells (Fig. 4B; Supplementary Fig. S4C). These changes correlated with lower p65 phosphorylation in TNF-treated HEY-CIP2AshRNA cells (Fig. 4C and D). To dissect at which level of the NF-κB pathway CIP2A confers its effects, we studied NF-κB promoter activity in combination with overexpression of the p65 upstream kinase MEKK3 (33). MEKK3 overexpression strongly induced NF-κB promoter activity in HEY-CTLshRNA cells, but this was blunted in HEY-CIP2AshRNA cells (Fig. 4E; lane 3 vs. 4). However, CIP2A inhibition was able to blunt NF-κB activity also in cells overexpressing MEK mutant with nondephoshorylatable serine 250 and threonine 516 (MEKK3S250D/T516D; ref. 34; Fig. 4E; lane 7 vs. 8). These findings together with CIP2A effects on p65 phosphorylation (Fig. 4C, D), support the conclusions that CIP2A promotes NF-κB activity downstream of activated MEKK3.

Figure 4.

CIP2A promotes NF-κB activity in APR-246–insensitive ovarian cancer cells. A, Relative NF-κB luciferase reporter activity in HEY-CTLshRNA and HEY-CIP2AshRNA cells. Shown is mean ± SEM. *, P ≤ 0.05, Mann–Whitney test. B, Quantification of p65 signal intensity ratio (Nuclear/Cytoplasm) in HEY-CTLshRNA or HEY-CIP2AshRNA cells with or without TNFα treatment. Shown is mean ± SEM. ***, P ≤ 0.001, Mann–Whitney test. C, Representative Western blot analysis of phospho-P65 and total p65 from TNF-α–treated HEY-CTLshRNA or HEY-CIP2AshRNA1 and HEY-CIP2AshRNA2 cells. D, Quantification of relative p65 phosphorylation between HEY-CTLshRNA or HEY-CIP2AshRNA1 cells from five independent experiments as C. ***, P ≤ 0.05, Mann–Whitney test. E, Relative NF-κB luciferase reporter activity in HEY-CTLshRNA or HEY-CIP2AshRNA cells with either empty vector, MEKK3 WT, MEKK3 T516A/S250A, or MEKK3 T5163/S250D overexpression. Shown is mean ± SEM. **, P ≤ 0.01; ***, P ≤ 0.001, Mann–Whitney test. F and G, Relative cell viability of HEY-CTLshRNA cells treated with APR-246 alone, or with IKK inhibitors PS-1145 or BMS-345541 alone, and their combinations. ***, P ≤ 0.001, t test. H, Colony growth assay of HEY-CTLshRNA, HEY-CIP2AshRNA1, and HEY-CIP2AshRNA2 treated with either vehicle, PS-1145 alone, APR-246 alone, or PS-1145 + APR-246. I, Relative cell viability in patient-derived HGSC cell line OC002 treated with either APR-246 alone or BMS-345541 + APR-246. EC50 values for APR-246 in each condition are indicated next to concentration curve. J, Model of mechanistic basis of CIP2A-mediated APR-246 resistance in ovarian cancer cells. Grey denotes for inhibited status of the indicated protein.

Figure 4.

CIP2A promotes NF-κB activity in APR-246–insensitive ovarian cancer cells. A, Relative NF-κB luciferase reporter activity in HEY-CTLshRNA and HEY-CIP2AshRNA cells. Shown is mean ± SEM. *, P ≤ 0.05, Mann–Whitney test. B, Quantification of p65 signal intensity ratio (Nuclear/Cytoplasm) in HEY-CTLshRNA or HEY-CIP2AshRNA cells with or without TNFα treatment. Shown is mean ± SEM. ***, P ≤ 0.001, Mann–Whitney test. C, Representative Western blot analysis of phospho-P65 and total p65 from TNF-α–treated HEY-CTLshRNA or HEY-CIP2AshRNA1 and HEY-CIP2AshRNA2 cells. D, Quantification of relative p65 phosphorylation between HEY-CTLshRNA or HEY-CIP2AshRNA1 cells from five independent experiments as C. ***, P ≤ 0.05, Mann–Whitney test. E, Relative NF-κB luciferase reporter activity in HEY-CTLshRNA or HEY-CIP2AshRNA cells with either empty vector, MEKK3 WT, MEKK3 T516A/S250A, or MEKK3 T5163/S250D overexpression. Shown is mean ± SEM. **, P ≤ 0.01; ***, P ≤ 0.001, Mann–Whitney test. F and G, Relative cell viability of HEY-CTLshRNA cells treated with APR-246 alone, or with IKK inhibitors PS-1145 or BMS-345541 alone, and their combinations. ***, P ≤ 0.001, t test. H, Colony growth assay of HEY-CTLshRNA, HEY-CIP2AshRNA1, and HEY-CIP2AshRNA2 treated with either vehicle, PS-1145 alone, APR-246 alone, or PS-1145 + APR-246. I, Relative cell viability in patient-derived HGSC cell line OC002 treated with either APR-246 alone or BMS-345541 + APR-246. EC50 values for APR-246 in each condition are indicated next to concentration curve. J, Model of mechanistic basis of CIP2A-mediated APR-246 resistance in ovarian cancer cells. Grey denotes for inhibited status of the indicated protein.

Close modal

To address whether CIP2A-driven NF-κB activity functionally confers APR-246 resistance, we tested whether similar synergy that was observed between CIP2A inhibition and APR-246, could be recapitulated by cotreatment of HEY-CTLshRNA cells with APR-246 and small-molecule inhibitors of NF-κB. As a result, all three tested NF-κB inhibitors, each with different mode of action, potentiated the effect of APR-246 in inhibition of cell viability in HEY-CTLshRNA cells (Fig. 4F and G; Supplementary Fig. S4D). These results were substantiated by colony growth assays including two independent HEY-CIP2A shRNA cell clones with different CIP2A shRNAs. With the chosen dose, NF-κB inhibitor PS-1145 did not have any notable effect on either HEY-CTLshRNA or HEY-CIP2AshRNA cells, but in combination with APR-246, it induced a similar synthetic lethal phenotype that was observed with APR-246 in HEY-CIP2AshRNA cells (Fig. 4H). Finally, the combined action of APR-246 and NF-κB inhibition was validated in patient-derived ovarian cancer cell line OC002 derived from a patient with disseminated disease (Fig. 4I; ref. 11). These results indicate that inhibition of NF-κB activity mediates APR-246 sensitivity in CIP2A-depleted ovarian cancer cells (Fig. 4J). This can be owed to higher PP2A activity in CIP2A-deficient cells leading to dephosphorylation of RelA and inactivation of the NF-κB downstream signaling (35, 36).

During relapse from chemotherapy, the ovarian cancer cells have exhausted their capacity to respond to DNA-damaging agents (1) but might yet be vulnerable to other therapies in a subtype-specific manner (2). Our results collectively identify CIP2A as a context-dependent oncoprotein in ovarian cancer. It is dispensable for both human and mouse ovarian cancer tumorigenesis, but associates with more aggressive disease (ref. 8; Supplementary Table S1), and drives resistance to APR-246 therapy. Together with our analysis from a limited number of available human relapse samples, these data indicate that ovarian cancer tumors with low CIP2A expression constitute a minor but yet clinically relevant human ovarian cancer subtype. Combined with the recently demonstrated role of CIP2A in confining therapy response for dozens of commonly used cancer drugs in other cancer cell types (12, 13), our results encourage further screening of CIP2A-depleted ovarian cancer cell models against larger drug libraries to identify additional drugs to be tested for the treatment of CIP2A-deficient ovarian cancer subtype patients.

Current data indicate that APR-246 kills cancer cells via multiple mechanisms (20, 21, 24–26). Our data on the ROS-dependent, but most likely TP53-independent mechanism of ovarian cancer cell killing by APR-246 is directly supported by recently published work (21, 25). This is a potentially clinically important finding as it indicates that the TP53 status would not dictate the cell killing activity of APR-246 in CIP2A-deficient ovarian cancer subtype tumors. APR-246 has been tested in two ovarian cancer clinical trials (NCT02098343, NCT03268382) but no results are publicly available. Currently, APR-246 is studied in clinical trials in AML and myelodysplastic syndromes (22), and in various solid cancer types (https://www.clinicaltrials.gov). Similar to ovarian cancer, also among these cancer types there are a significant number of patients with CIP2A-deficient subtype (4, 7). Therefore, and acknowledging the role of NF-κB activity in the regulation of cellular buffering capacity against ROS (29), it would be very interesting to examine the CIP2A expression and NF-κB pathway activity from clinical trial patient samples from these past and ongoing APR-246 trials. By these means, the presented results could support a better prediction of the potential responders in future APR-246 clinical trials and thus establish a future patient stratification strategy for the clinical use of APR-246. Furthermore, based on the initial screening results (Fig. 1A), hypersensitivity of CIP2A-deficient cells to ROS-inducing drugs might not be unique to APR-246. Although APR-246 had the greatest CIP2A-dependent effect, other ROS-inducing drugs such as cisplatin, paclitaxel, and doxorubicin also had increased potency in CIP2A-deficient cells (Fig 1A). This warrants further investigation into the effect of ROS-inducing drugs on CIP2A-deficient cells as a plausible strategy for identification of subpopulations of patients with increased sensitivity to targeted therapies. Finally, our results position PP2A inhibitor protein CIP2A as an APR-246 combination therapy target in ovarian cancer. In that regard, a recent study showed that small molecular activators of PP2A (SMAPs; ref. 37) activate PP2A at least partly via inhibition of CIP2A expression (17). In light of these results, combination of APR-246 (or other ROS-inducing drugs) with future derivatives of SMAPs could be a possible treatment for patients with ovarian cancer with high tumor CIP2A expression.

U. Butt reports nonfinancial support from Turku, Åbo Akademi University, and Biocenter Finland during the conduct of the study. H. Lassus reports grants from Helsinki University Hospital during the conduct of the study and personal fees from Astra Zeneca, GlaxoSmithKline, and Eisai outside the submitted work. D.C. Connolly reports grants from NCI CA006927 during the conduct of the study and has a patent for US 17/430,227 pending. J. Pouwels reports grants from the Academy of Finland during the conduct of the study. J. Westermarck reports grants from Sigrid Juselius Foundation and Finnish Cancer Foundation; nonfinancial support from APREA during the conduct of the study; and other support from MERCK GmbH and ORION Pharma outside the submitted work. No disclosures were reported by the other authors.

A.N. Cvrljevic: Conceptualization, formal analysis, investigation. U. Butt: Formal analysis, investigation, visualization, writing–original draft, writing–review and editing. K. Huhtinen: Resources, investigation. T.J. Grönroos: Formal analysis, investigation. C. Böckelman: Formal analysis. H. Lassus: Data curation, formal analysis. R. Butzow: Resources. C. Haglund: Resources. K. Kaipio: Investigation. T. Arsiola: Visualization, project administration, writing–review and editing. T.D. Laajala: Data curation, software. D.C. Connolly: Resources. A. Ristimäki: Resources. O. Carpen: Resources. J. Pouwels: Conceptualization, supervision, investigation. J. Westermarck: Conceptualization, resources, supervision, funding acquisition, writing–original draft, project administration.

We are very grateful to APREA Therapeutics for APR-246 compound and Dr. Jianhua Yang for MEKK3 plasmids. This study used Turku Bioscience Centre core services by Finnish Functional Genomics Centre, and Cell Imaging and Cytometry, funded by University of Turku and Åbo Akademi University and Biocenter Finland. Turku Center for Disease Modeling (TCDM), funded by University of Turku and Biocenter Finland is acknowledged for expert assistance with the animal experiments. Fox Chase Cancer Center (FCCC) is acknowledged for the husbandry and exportation of the TgMISIIR-TAg mice used in this study. The work was funded by Sigrid Juselius Foundation (to J. Westermarck), Finnish Cancer Foundation (to J. Westermarck), Helsinki University Central Hospital Research Funds (to A. Ristimäki), Finska Läkaresällskapet (to A. Ristimäki) and Fox Chase Cancer Center (FCCC) Core Grant NCI P30 CA006927 (to D.C. Connolly). D.C. Connolly was also funded with generous donations from the Dubrow Fund and the Bucks County Board of Associates and the Mainline Board of Associates. T.D. Laajala was funded as an FICAN Cancer Researcher for the Finnish Cancer Institute and by the Finnish Cultural Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data