Reactivation of p53 tumor-suppressor function by small molecules is an attractive strategy to defeat cancer. A potent p53-reactivating molecule RITA, which triggers p53-dependent apoptosis in human tumor cells in vitro and in vivo, exhibits p53-independent cytotoxicity due to modifications by detoxification enzyme Sulfotransferase 1A1 (SULT1A1), producing a reactive carbocation. Several synthetic modifications to RITA's heterocyclic scaffold lead to higher energy barriers for carbocation formation. In this study, we addressed the question whether RITA analogs NSC777196 and NSC782846 can induce p53-dependent apoptosis without SULT1A1-dependent DNA damage. We found that RITA analog NSC782846, but not NSC777196, induced p53-regulated genes, targeted oncogene addiction, and killed cancer cells upon p53 reactivation, but without induction of DNA damage and inhibition RNA pol II. Our results might demonstrate a method for designing more specific and potent RITA analogs to accelerate translation of p53-targeting compounds from laboratory bench to clinic.

The tumor-suppressor p53 is a transcription factor that binds to the promoters of its target genes and regulates their expression. Upon DNA damage and other types of stresses, p53 blocks proliferation of pre-malignant and malignant cells or eliminates them by inducing apoptosis (1–3). p53 inactivation via mutations or enhanced degradation by MDM2 is the most frequent alteration in human cancers, which underscores the key role of p53 in combating cancer (4–6). Reinstatement of p53 by genetic means has demonstrated remarkable tumor suppression in animal models, including inhibition of aggressive metastatic lesions (7–10). This inspires the idea of developing small molecules reactivating p53 to fight cancer.

We have identified a small-molecule RITA (reactivation of p53 and induction of tumor cell apoptosis), which prevents p53/MDM2 interaction, induces p53 accumulation and transcriptional activity, and triggers p53-dependent apoptosis in tumor cells of a different origin in vitro and in mice (11–13). However, at levels similar to efficacious doses observed in mouse xenografts of human cancer cell lines, RITA caused pulmonary edema in rat, dog, and monkey (14), but not in mice, which are deficient for the expression of phenol sulfotransferase detoxification enzymes in lung (15). We found that p53-independent cytotoxicity of RITA requires modification by SULT1A1 (16). Hence, further development of this chemotype necessitates the generation of analogs for which SULT1A1 expression is not sufficient for p53-independent activity.

With the development of precision medicine that specifically targets cancer drivers, such as imatinib (Gleevec) and others, these types of therapeutics can be used in the clinic with fewer adverse side effects (17–19). The concept of selectively targeting specific types of cancer cells, rather than generating broadly cytotoxic compounds, is now widely viewed as a desirable route for drug development. With respect to RITA, we hypothesize that broad toxicity observed in multiple mammals was due to nonspecific biological activities of the SULT1A1-modified molecule, such as induction of DNA damage and inhibition of transcription processes (RNA polymerase II stalling and degradation; ref. 20). More target-selective analogs, recently designed by Developmental Therapeutics Program (DTP) at National Cancer Institute (NCI), in collaboration with Peyser and colleagues, narrowed the cytotoxicity profile of RITA without elimination of in vivo antitumor activity.

In this study, we applied multidimensional approaches, including analysis of gene expression profiles, analysis of NCI pharmacology database and functional assays, to identify the mechanisms of action (MoA) of RITA analogs NSC777196 and NSC782846. These findings might indicate how to design novel highly selective p53-targeting compounds.

Cell lines

MCF7, HCT116 (NCI-DTP Cat# HCT-116, RRID:CVCL_0291), T47D, A375, H1299, A431, and MDA-MB-231 (RRID:CVCL_ZZ22) were cultured in DMEM supplemented with 10% FBS (Gibco), 100 U/mL of penicillin, and 100 mg/mL of streptomycin (Sigma-Aldrich). SJSA and U2OS (kindly provided by Lars-Gunnar Larsson, Karolinska Institutet) were cultured in RPMI-1640 with 10% FBS and antibiotics. CRISPR/Cas9-mediated SULT1A1 deletion was performed in MCF7 and HCT116 cells using sgRNA-targeting exon 4 and exon 7. The SJSA-1 and A375 cells were transduced with TP53gRNA lentiviruses and p53 KO cells were enriched using 10 μmol/L of Nutlin treatment as described previously (21). CRISPR/Cas9-mediated p53 deletion was performed in MCF7 cells using sgRNA-targeting TP53 exon 4. A375 and SJSA cells, stably expressing SULT1A1 cDNA (OriGene, #RC201601L1), were generated by lentivirus transduction as described previously (16). Cell lines were authenticated by ATCC and Mycoplasma contamination was tested monthly using the MycoAlert Mycoplasma Detection Kit (Lonza) according to the manufacturer's instructions. All experiments were performed within 6 passages from frozen stocks.

Compounds

RITA (NSC652287) was obtained from the NCI. NSC777196 and NSC782846 were a generous gift from Dr. P. Wipf. DMSO, ActD (actinomycin D), 4OH-TMX and Nutlin-3 were purchased from Sigma-Aldrich. The compound concentrations and durations of treatment are mentioned in the figure legends.

Western blotting

To prepare protein lysates, cells were harvested, washed, and lysed in ice-cold RIPA buffer [150 mmol/L NaCl, 5 mmol/L Tris (pH 8.0), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS] supplemented with complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitors (Roche). Specific antibodies used for endogenous protein detection by Western blot in this study are the following:

IGF1-R (#9750, Cell Signaling Technology), MCL1 (Santa Cruz Biotechnology Cat# sc-819, RRID:AB_2144105), c-Myc (ab205818, Abcam), Bcl-2 (sc-7382, Santa Cruz Biotechnology), MDM2 (337100, Life technologies), MDMX (A300–287B, Bethyl Lab), p21 (#610233, BD Transduction), NOXA (114C307, Calbiochem), p53 (Santa Cruz Biotechnology Cat# sc-126, RRID:AB_628082), PARP (#95423, Cell Signaling Technology), phosphoS2-RNA polymerase II (ab5095, Abcam), RNA pol II (#05–623, Millipore), γH2AX (#07–164, Millipore), and SULT1A1 (ab191069, Abcam). Anti–β-actin mAb (MAB1501, Millipore) was used as loading control. Densitometric quantification of the bands was performed using ImageJ software (ImageJ, RRID:SCR_003070) and normalized to β-actin level.

qPCR

Total RNA extraction and cDNA synthesis were performed using Aurum total RNA and iScript cDNA Synthesis Kits (Bio-Rad) according to the manufacturer's instructions. mRNA quantification was performed by qRT-PCR using Sso Advanced Universal SYBRGreen SuperMix (Bio-Rad). RPLPO and RPL13A were used as housekeeping genes. Error bars represent SD from mean of at least three independent experiments. The sequences of qPCR primers used are detailed in Supplementary Table S1.

Correlation sensitivity analysis

NCI-60 mRNA expression values were obtained from Affymetrix HG-U133 Plus 2 microarrays with NCBI GEO accession number GSE32474. The data were gcrma-normalized using the justGCRMA function and the optimize.by = “memory” option in R/Bioconductor version 3.6, and then each probe set was summarized using the median log2 expression value within each cell line. Regression analyses were performed with linear models in R version 4.1, and the adjusted R2 value was used to estimate the 50% GI50 variance accounted for by log2 mRNA expression.

Cell viability assays and synergy analysis

Cell viability was assessed by resazurin assay. Briefly, cells were treated for 24–72 hours and incubated for 2 hours with 5 μmol/L resazurin (Sigma-Aldrich) before fluorescence measurement on a FLUOstar Omega (BMG LABTECH). For crystal violet staining experiments, cells were treated for 3–5 days, fixed with 3.7% paraformaldehyde, and stained with 0.2% crystal violet (Sigma-Aldrich).

To determine the dose-dependent effects of combination therapy, the Chou–Talalay method and CalcuSyn software (version 2) were used (22). Interaction between NSC782846 and 4-OH tamoxifen was quantified on the basis of a combination index (CI) to assess synergism (CI < 1), additive effect (CI = 1), and antagonism (CI > 1).

Lentivirus-mediated p53 knockdown

Lentiviruses containing TP53 shRNA#1 and TP53 shRNA#2 to knockdown p53 were transduced with Polybrene (Sigma-Aldrich#H9268) for 24 hours before treatment. Scrambled shRNA and shRNA-targeting GFP (a kind gift from Dr. Margareta Wilhelm, Karolinska Institutet) were used as negative control. All shRNA sequences are listed in Supplementary Table S1.

5-Ethynyluridine labeling

Cells were treated on glass coverslips in 12-well plates for the indicated time, and the medium was changed to the one containing 1 mmol/L 5-ethynyluridine (EU) combined with original treatment 1 hour before cell fixation. EU was labeled using the Click-iT RNA Alexa Fluor 594 Imaging Kit (Thermo Fisher Scientific) according to the manufacturer's instructions.

Immunofluorescence

For immunostaining, 50k cells were plated on glass coverslips in 12-well plates the day before the experiment. After treatment, cells were fixed in 4% paraformaldehyde for 15 minutes, permeabilized in PBS/0.1%TritonX-100 for 15 minutes at room temperature (RT) and blocked in 5% FBS in PBS (blocking buffer) for 30 minutes at RT. Cells were incubated in primary antibody diluted in blocking buffer overnight at 4°C, washed three times in PBS and incubated with Alexa-conjugated secondary antibodies (Life technologies) diluted in PBS for 1 hour at RT. Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific). After washing 3 times in PBS cells were mounted in ProLong Gold Antifade Reagent (Life technologies). Primary antibodies against p53 (sc126, Santa Cruz Biotechnology) and γH2AX (#07–164, Millipore) were used for immunostainings. Images were taken with a ZEISS fluorescence microscope.

Flow cytometry

Apoptosis measured by Annexin V-FITC/propidium iodide (PI) staining. An Annexin V-FITC/PI apoptosis kit (Invitrogen) was used to quantify the percentage of cells undergoing apoptosis, according to the manufacturer's instructions. BD FACS Canto II flow cytometer was used to detect apoptotic cells. Cell population in different quadrants was calculated statistically.

The online database

cBioPortal for Cancer Genomics (RRID:SCR_014555 http://www.cbioportal.org/; refs. 23, 24) was used to analyze the correlation of mutations related to breast cancer ER status. Detailed information of breast cancer patients is shown in Supplementary Tables S2 and S3.

Statistical analysis

The relationship between ER Status, MDM2 gene amplification, and MDM4 gene amplification was analyzed using a χ2 test. Unless otherwise stated, statistical significance was calculated using a two-tailed Student t test.

Data availability

The data generated in this study are available upon request from the corresponding author.

The antitumor activity of RITA analogs shows decreased SULT1A1 dependence

We investigated how a series of synthetic modifications to RITA heterocyclic scaffold in NSC777196 and NSC782846 (Fig. 1A) affect their SULT1A1 dependence. Analysis of the NCI-60 pharmacological database revealed a decreased correlation between the baseline expression level of SULT1A1 and cell growth inhibition by RITA analogs compared with RITA (R2 = 0.62 for RITA; R2 = 0.45 for NSC777196; R2 = 0.43 for NSC782846; Fig. 1B).

Figure 1.

Anticancer activity of RITA, NSC777196, and NSC782846. A, Chemical structures of RITA, NSC777196, and NSC782846. B, Correlation of SULT1A1 mRNA level with sensitivity to RITA, NSC777196, and NSC782846 in NCI-60 cell line database. C, Top, SULT1A1 protein level detected by Western blot in seven cancer cell lines, including breast carcinoma T47D, MDAMB-231; skin cancer A431; osteosarcoma U2OS, SJSA; melanoma A375; and lung carcinoma H1299. β-Actin was used as loading control. Bottom, NSC777196 efficiently suppressed the growth of high SULT1A1-expressing cells (U2OS, T47D, and A431, red lines) while having negligible effect in cells with low SULT1A1 (A375, MDAMB-231, SJSA, H1299, black lines). The antitumor effect of NSC782846 was less dependent on the level of SULT1A1 expression. Shown are the results obtained from five biological replicates. D, Cell viability of WT and SULT1A1−/− MCF7 cells after 24 hours treatment with RITA, NSC777196, and NSC782846 (n = 5; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 1.

Anticancer activity of RITA, NSC777196, and NSC782846. A, Chemical structures of RITA, NSC777196, and NSC782846. B, Correlation of SULT1A1 mRNA level with sensitivity to RITA, NSC777196, and NSC782846 in NCI-60 cell line database. C, Top, SULT1A1 protein level detected by Western blot in seven cancer cell lines, including breast carcinoma T47D, MDAMB-231; skin cancer A431; osteosarcoma U2OS, SJSA; melanoma A375; and lung carcinoma H1299. β-Actin was used as loading control. Bottom, NSC777196 efficiently suppressed the growth of high SULT1A1-expressing cells (U2OS, T47D, and A431, red lines) while having negligible effect in cells with low SULT1A1 (A375, MDAMB-231, SJSA, H1299, black lines). The antitumor effect of NSC782846 was less dependent on the level of SULT1A1 expression. Shown are the results obtained from five biological replicates. D, Cell viability of WT and SULT1A1−/− MCF7 cells after 24 hours treatment with RITA, NSC777196, and NSC782846 (n = 5; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

Next, we validated the decreased SULT1A1 dependence of anticancer activities by RITA analogs. We assessed the effects of NSC777196 and NSC782846 in seven cancer cell lines with different SULT1A1 levels (Fig. 1C, top). We performed proliferation assay in cell lines with low SULT1A1: wtp53 osteosarcoma SJSA, mutp53 breast carcinoma MDA-MB231, wtp53 melanoma A375, p53-null lung adenocarcinoma H1299; and with high SULT1A1: mutp53 breast carcinoma T47D, mutp53 epidermoid carcinoma A431, and wtp53 osteosarcoma U2OS.The level of SULT1A protein is shown in the top in Fig. 1C and the growth suppression by NSC782846 and NSC777196 is shown in the bottom in Fig. 1C. As evident from Fig. 1C, the response to NSC782846 was much less dependent on SULT1A1 than that of NSC777196.

To further validate the decreased SULT1A1 dependence of growth suppression by RITA analogs, we performed CRISPR–Cas9-mediated deletion of SULT1A1 alleles in MCF7 and HCT116 cell lines (Supplementary Fig. S1A). Although RITA and NSC777196 inhibited cell proliferation in a SULT1A1-dependent manner, NSC777196 was more efficient in the MCF7 SULT1A1 knock out (KO) cell line than RITA. The viability of MCF7 SULT1A1KO cells reached 73% by NSC777196 compared with 82% by RITA, whereas the same dose leads to wild-type (WT) cell line reduction to 49% (Fig. 1D, left and middle). The growth suppression by NSC782846 was similar in the MCF7 SULT1A1KO and WT cell lines when the concentration was higher than 10 μmol/L (Fig. 1D, right), suggesting a decreased dependence of growth suppression on SULT1A1. In the HCT116 cell line, all three compounds demonstrated dependence on SULT1A1 expression, albeit to a different extent. HCT116 SULT1A1–/– was resistant to RITA and NSC777196 at the GI50 dose in the HCT116 WT cell line, whereas NSC782846 resulted in 8% cell death (Supplementary Fig. S1B).

In addition to the engineered SULT1A1 deletion by CRISPR–Cas9 in HCT116 and MCF7 cell models, we tested the dependence on SULT1A using two additional cell models. We engineered SULT1A1 status in wtp53 osteosarcoma SJSA and melanoma A375 with low SULT1A1, in which we overexpressed SULT1A1 using lentivirus (Supplementary Fig. S1C). As shown in the top in Supplementary Fig. S1D, overexpression of SULT1A1 in SJSA cells did not change the response to NSC782846, whereas it sensitized to RITA and NSC777196. In A375 cells overexpression of SULT1A1 significantly increased sensitivity to RITA and NSC777196, whereas the effect on response to NSC782846 was much less pronounced (Supplementary Fig. S1D, bottom).

Overall, these data show that the decreased SULT1A1 dependence of the growth suppression by RITA analogs, NSC782846 in particular.

Transcriptional repression of oncogenes and growth suppression of RITA analogs are p53-dependent

We confirmed the induction of the p53 protein levels by RITA analogs using immunofluorescence staining (Supplementary Fig. S2A). To explore the effects of restoring p53 function in tumor cells by NSC777196 and NSC782846, we analyzed the changes in gene expression in isogenic MCF7 WT and MCF7 p53KO cells after treatment by using qPCR. We observed a marked downregulation of representative oncogenes: IGF1R, MYC (c-Myc) and MCL1 (Fig. 2A), in line with our previous data obtained upon treatment with RITA (13). Transcriptional repression of oncogenes was p53-dependent, as we observed much less or no changes in the expression of these genes in the p53KO counterpart cell line by NSC777196 and NSC782846, respectively (Fig. 2A). Consistent with the mRNA level, the protein level of oncogenes was significantly decreased after treatment. Inhibition of oncogenes was partly or totally rescued in the p53KO cell line upon NSC777196 and NSC782846 treatment, respectively (Fig. 2B; Supplementary Fig. S2B), corroborating p53 dependence. We further confirmed this result in another cell line, HCT116 (Supplementary Fig. S2C).

Figure 2.

RITA analogs repress transcription of oncogenes and suppress cancer cells growth in a p53-dependent manner. A, mRNA levels of oncogenes were detected by qPCR in MCF7 and MCF7 p53–/– cells 6 hours after treatment with 5 μmol/L NSC777196 and 10 μmol/L NSC782846. B, Protein levels of selected oncogenes in MCF7 and MCF7 p53–/– cells upon 6 hours treatment by 5 μmol/L NSC777196 and 10 μmol/L NSC782846 as detected by Western blotting. Quantification of protein levels is indicated below. C, Cell viability of WT and p53−/− MCF7 cells after 24 hours treatment with NSC782846 (n = 5; *, P < 0.05; **, P < 0.01). D, Crystal violet staining of MCF7 and MCF7 p53–/− cells after 3 days of treatment with NSC782846.

Figure 2.

RITA analogs repress transcription of oncogenes and suppress cancer cells growth in a p53-dependent manner. A, mRNA levels of oncogenes were detected by qPCR in MCF7 and MCF7 p53–/– cells 6 hours after treatment with 5 μmol/L NSC777196 and 10 μmol/L NSC782846. B, Protein levels of selected oncogenes in MCF7 and MCF7 p53–/– cells upon 6 hours treatment by 5 μmol/L NSC777196 and 10 μmol/L NSC782846 as detected by Western blotting. Quantification of protein levels is indicated below. C, Cell viability of WT and p53−/− MCF7 cells after 24 hours treatment with NSC782846 (n = 5; *, P < 0.05; **, P < 0.01). D, Crystal violet staining of MCF7 and MCF7 p53–/− cells after 3 days of treatment with NSC782846.

Close modal

Because the effects of NSC782846 on oncogene inhibition were totally p53-dependent, we tested whether inhibition of cell proliferation was also p53-dependent. Indeed, using short-term and long-term assays, we found a significant contribution of p53 to cancer cell growth, suppression upon NSC782846 (Fig. 2C; for 48 and 72 hours time point see Supplementary Fig. S2D). We found that the role of p53 in growth suppression by NSC777196 was quite weak, although detectable (Supplementary Fig. S2D). We confirmed this result in another two cell lines, A375 and SJSA (Supplementary Fig. S2E–S2G). Our results obtained in short-term and long-term growth suppression assays shown in Supplementary Fig. S2F and S2G corroborated the p53-dependent effect of NSC782846 in A375 and SJSA cell lines, because p53 KO by CRISPR–Cas9 significantly prevented growth inhibition. In addition, the effect of treatment with NSC782846 was irreversible, because it was evident upon washing out the compound after 48 hours of treatment (Supplementary Fig. S2G).

In summary, our results show that NSC782846 treatment leads to downregulation of oncogenes expression in a p53-dependent manner. p53 contribution to growth suppression by NSC782846 is significant, whereas the role of p53 in the effects of NSC777196 is less substantial.

Dose-dependent repression of oncogenes by RITA analogs

Our results suggest that pharmacologically reactivated p53 acts as a potent repressor of a number of oncogenic and survival factors, as well as functions as a powerful trigger of p53 targets, pro-apoptotic NOXA and cdk inhibitor p21 (Fig. 3A). Furthermore, we found that the transactivation of p53 target genes requires a lower dose of NSC777196 than transrepression of pro-survival genes. As evident from Fig. 3A, left, in response to 0.5 μmol/L NSC777196 p53 and its targets NOXA and p21 were induced. In contrast, oncogenes were regulated differently, whereas 5 μmol/L NSC777196 was sufficient to trigger a sharp downregulation of c-Myc, MCL1, MDM2, and Bcl-2, upon treatment with 0.5 μmol/L NSC777196 the decline of these oncogenes was less pronounced.

Figure 3.

Dose-dependent repression of oncogenes by RITA analogs. A, Levels of pro-apoptotic factors NOXA and representative survival factors c-Myc, MCL1, MDM2, IGF1-R, and Bcl-2 upon 0.5 and 5 μmol/L NSC777196 were detected by Western blotting (left). Levels of proteins mentioned above after 12 and 24 hours of NSC782846 treatment of MCF7 cells were detected by Western blotting (right). B, mRNA levels of MCL1, MYC, IGF1R, MDM2, BCL-2, CDKN1A, BBC3, and PMAIP after 16 hours of treatment with 5 μmol/L NSC777196 or 10 μmol/L NSC782846 as detected by qPCR (n = 3 *, P < 0.05; **, P < 0.01; ***, P < 0.001). C, Apoptotic MCF7 cells were detected by Annexin V-PI double staining using FACS after treatment for 16 hours with 5 μmol/L NSC777196 or 10 μmol/L NSC782846. Representative pictures are shown in left. The total number of cells in the Q2 and Q3 quadrant was regarded as apoptotic cells. Percentages of apoptotic cells are shown in the bar graph (right; n = 3 **, P < 0.01).

Figure 3.

Dose-dependent repression of oncogenes by RITA analogs. A, Levels of pro-apoptotic factors NOXA and representative survival factors c-Myc, MCL1, MDM2, IGF1-R, and Bcl-2 upon 0.5 and 5 μmol/L NSC777196 were detected by Western blotting (left). Levels of proteins mentioned above after 12 and 24 hours of NSC782846 treatment of MCF7 cells were detected by Western blotting (right). B, mRNA levels of MCL1, MYC, IGF1R, MDM2, BCL-2, CDKN1A, BBC3, and PMAIP after 16 hours of treatment with 5 μmol/L NSC777196 or 10 μmol/L NSC782846 as detected by qPCR (n = 3 *, P < 0.05; **, P < 0.01; ***, P < 0.001). C, Apoptotic MCF7 cells were detected by Annexin V-PI double staining using FACS after treatment for 16 hours with 5 μmol/L NSC777196 or 10 μmol/L NSC782846. Representative pictures are shown in left. The total number of cells in the Q2 and Q3 quadrant was regarded as apoptotic cells. Percentages of apoptotic cells are shown in the bar graph (right; n = 3 **, P < 0.01).

Close modal

As for NSC782846, time but not dose plays an important role in downregulation of the oncogenic proteins (Fig. 3A, right). Upon either 5 or 10 μmol/L NSC782846 treatment for 12 hours the repression of c-Myc, MCL1, and IGF1-R was quite weak. Prolonged to 24 hours treatment produced a significant downregulation of oncogenes mentioned above. RITA as a positive control was much more efficient in oncogenes inhibition. p21 mRNA was induced, but its protein level was downregulated by RITA, as we have shown previously (25). In contrast, NSC782846 induced p21 both on mRNA and protein level that suggests a different MoA compared with RITA.

qPCR confirmed the transcriptional repression of IGF1R, MCL1, MYC, and BCL-2 and transcriptional activation of p53-target genes CDKN1A (encoding p21), BBC3 (PUMA) and PMAIP1 (NOXA) by both RITA analogs (Fig. 3B).

We used PI and Annexin-V double staining to detect apoptotic cells upon RITA analogs 16 hours post-treatment. As shown in Fig. 3C, both analogs can induce efficient apoptosis, but the fraction of apoptotic cells upon NSC782846 treatment was lower than upon NSC777196 treatment.

Taken together, our data imply that repression of oncogenes by NSC782846 occurs through a different mechanism compared with RITA and its analog NSC777196.

RITA analogs can induce p53 activation, p53-dependent induction of proapoptotic genes, repression of oncogenes and cancer cell apoptosis in SULT1A1-independent manner

Previous data have shown that RITA is modified by enzyme SULT1A1 and that its cancer cell cytotoxicity can be rescued by phenol sulfotransferase inhibition (14). To explore whether RITA analogs can inhibit cancer cell proliferation in the absence of SULT1A1, we analyzed their effects in the MCF7 SULT1A1–/– cell line.

We found that in the absence of SULT1A1 both compounds induced p53 accumulation, demonstrating that p53 activation is independent of SULT1A1. Along with p53 induction, treatment with RITA analogs lead to downregulation of several crucial oncogenes, IGF1-R, c-Myc, and MCL1, both on mRNA and protein level (Fig. 4A and B), similar to the downregulation of oncogenes we detected in SULT1A1-expressing cells (Fig. 2A and B). Besides oncogene repression, we detected the induction of p53 target genes in SULT1A1-deficient cells: proapoptotic PMAIP1 and BBC3, and cell-cycle arrest gene CDKN1A, although the kinetics of induction of these genes differed (Supplementary Fig. S3A).

Figure 4.

RITA analogs-induced cancer cell apoptosis correlates with ablation of oncogenes upon p53 reactivation. A, mRNA level of representative oncogenes after 16 hours treatment of MCF7 SULT1A1−/− cells with 10 μmol/L NSC777196 or 10 μmol/L NSC782846 measured by qPCR (n = 3 *, P < 0.05; **, P < 0.01). B, Downregulation of selected oncogenes upon treatment of MCF7 SULT1A1−/− cells by 10 μmol/L NSC777196 and 10 μmol/L NSC782846 as detected by Western blotting. C, Protein levels of selected oncogenes in MCF7 SULT1A1−/− in which p53 was depleted by 2 different shRNAs treated with 10 μmol/L NSC777196 or 10 μmol/L NSC782846 for 24 hours. Scrambled shRNA and GFP shRNA were used as controls. D, Apoptotic cells were detected by Annexin V-PI double staining using FACS after treatment of MCF7 SULT1A1 −/− cells with 10 μmol/L NSC777196 or 10 μmol/L NSC782846 for 16 hours. Representative pictures are shown in left. The total number of cells in the Q2 and Q3 quadrant was regarded as apoptotic cells. Percentages of apoptotic cells are shown in the bar graph (right; n = 3 *, P < 0.05).

Figure 4.

RITA analogs-induced cancer cell apoptosis correlates with ablation of oncogenes upon p53 reactivation. A, mRNA level of representative oncogenes after 16 hours treatment of MCF7 SULT1A1−/− cells with 10 μmol/L NSC777196 or 10 μmol/L NSC782846 measured by qPCR (n = 3 *, P < 0.05; **, P < 0.01). B, Downregulation of selected oncogenes upon treatment of MCF7 SULT1A1−/− cells by 10 μmol/L NSC777196 and 10 μmol/L NSC782846 as detected by Western blotting. C, Protein levels of selected oncogenes in MCF7 SULT1A1−/− in which p53 was depleted by 2 different shRNAs treated with 10 μmol/L NSC777196 or 10 μmol/L NSC782846 for 24 hours. Scrambled shRNA and GFP shRNA were used as controls. D, Apoptotic cells were detected by Annexin V-PI double staining using FACS after treatment of MCF7 SULT1A1 −/− cells with 10 μmol/L NSC777196 or 10 μmol/L NSC782846 for 16 hours. Representative pictures are shown in left. The total number of cells in the Q2 and Q3 quadrant was regarded as apoptotic cells. Percentages of apoptotic cells are shown in the bar graph (right; n = 3 *, P < 0.05).

Close modal

To further validate the p53-dependent and SULT1A1-independent inhibition of oncogenes, we depleted p53 by shRNA in MCF7 SULT1A1–/– cell line. Our results presented in Fig. 4C show that the downregulation of oncogenes was rescued after p53 knockdown in SULT1A1-depleted cells.

Notably, the greater oncogene downregulation upon 16 hours treatment with 10 μmol/L NSC782846 resulted in greater cell death compared with 10 μmol/L NSC777196 at the same time point (Fig. 4D), indicating that inhibition of oncogenes contributed to earlier apoptosis upon NSC782846 treatment. At extended treatment times, both RITA analogs led to apoptosis in the SULT1A1–/– cells, as detected by PARP cleavage (Supplementary Fig. S3B).

NSC782846 can induce p53 accumulation in the absence of DNA damage or transcriptional inhibition

We have previously shown that RITA treatment triggers well established p53 stimuli, DNA damage and transcriptional block via inhibition of RNA pol II (20). To find out whether RITA analogs can induce p53 in the absence of transcriptional inhibition and DNA damage, we used the MCF7 SULT1A1–/– cell line to perform the next series of experiments. In this cell line, RITA analogs are not modified by SULT1A1 to generate reactive carbocations, although we cannot rule out the possibility that other sulfotransferases can modify them. Notably, we found that in the absence of SULT1A1, NSC782846 did not induce the hallmark of DNA damage γH2AX (Fig. 5A and B; the densitometric quantification of the bands in Fig. 5A is shown in Supplementary Fig. S4A). However, p53 was strongly induced by NSC782846, suggesting a DNA damage-independent mechanism. On the other hand, NSC777196 leads to p53 accumulation along with induction of γH2AX, albeit weaker compared with RITA (Fig. 5A and B). These data suggest that although RITA and NSC777196 could be modified by sulfotransferases other than SULT1A1 (for example, SULT1A2 or SULT1E1), NSC782846 is less amenable to sulfonation.

Figure 5.

NSC782846 induces p53 accumulation without transcriptional inhibition or DNA damage. A, DNA damage marker γH2AX and p53 detected by Western blotting upon 24-hour treatment of MCF7 SULT1A1−/− cells with 1 μmol/L RITA, or 10 μmol/L and 25 μmol/L of NSC777196 or NSC782846, respectively. B, DNA damage marker γH2AX detected by immunostaining upon treatment by RITA (1 μmol/L), NSC777196 (10 μmol/L) and NSC782846 (10 μmol/L) in MCF7 SULT1A1−/− cells. C, RNA synthesis monitored by EU incorporation in MCF7 SULT1A1−/− cells treated with RITA (1 μmol/L), NSC777196 (10 μmol/L), NSC782846 (10 μmol/L), and ActD (2.5 mmol/L), then incubated for 1 hour with 1 mmol/L EU. EU was labeled by click chemistry and visualized by fluorescent microscopy. D, Protein levels of total RNA Pol II and phospho-Ser2 RNA Pol II in MCF7 SULT1A1−/− cells treated with RITA (1 μmol/L), NSC777196 (10 μmol/L), NSC782846 (10 μmol/L) for 24 hours. E, The levels of representative oncogenes and RNA Pol II upon treatment with low dose of NSC777196 and NSC782846 in MCF7 WT cells as detected by Western blotting. F, RNA synthesis monitored by EU incorporation in MCF7 WT cells treated with NSC777196 (0.25 μmol/L), NSC782846 (1 μmol/L), then incubated for 1 hour with 1 mmol/L EU. EU was labeled by click chemistry and visualized by fluorescent microscopy.

Figure 5.

NSC782846 induces p53 accumulation without transcriptional inhibition or DNA damage. A, DNA damage marker γH2AX and p53 detected by Western blotting upon 24-hour treatment of MCF7 SULT1A1−/− cells with 1 μmol/L RITA, or 10 μmol/L and 25 μmol/L of NSC777196 or NSC782846, respectively. B, DNA damage marker γH2AX detected by immunostaining upon treatment by RITA (1 μmol/L), NSC777196 (10 μmol/L) and NSC782846 (10 μmol/L) in MCF7 SULT1A1−/− cells. C, RNA synthesis monitored by EU incorporation in MCF7 SULT1A1−/− cells treated with RITA (1 μmol/L), NSC777196 (10 μmol/L), NSC782846 (10 μmol/L), and ActD (2.5 mmol/L), then incubated for 1 hour with 1 mmol/L EU. EU was labeled by click chemistry and visualized by fluorescent microscopy. D, Protein levels of total RNA Pol II and phospho-Ser2 RNA Pol II in MCF7 SULT1A1−/− cells treated with RITA (1 μmol/L), NSC777196 (10 μmol/L), NSC782846 (10 μmol/L) for 24 hours. E, The levels of representative oncogenes and RNA Pol II upon treatment with low dose of NSC777196 and NSC782846 in MCF7 WT cells as detected by Western blotting. F, RNA synthesis monitored by EU incorporation in MCF7 WT cells treated with NSC777196 (0.25 μmol/L), NSC782846 (1 μmol/L), then incubated for 1 hour with 1 mmol/L EU. EU was labeled by click chemistry and visualized by fluorescent microscopy.

Close modal

To investigate the effect of RITA analogs on transcriptional block, we visualized active transcription by following incorporation of EU into nascent RNA in MCF7 SULT1A1–/– cells. As shown in Fig. 5C, positive control RITA abolished RNA synthesis, similar to the known RNA Pol II inhibitor ActD. However, RITA analogs NSC777196 and NSC782846 did not block RNA synthesis. In line with these results, we found that the levels of total and phospho-S2 (elongating form) RNA Pol II were strongly decreased upon RITA treatment, but not upon treatment with RITA analogs (Fig. 5D). Further evidence for the crucial role of SUTL1A1-medited modification of RITA and its analogs in the inhibition of transcription comes from the results obtained upon overexpression of SULT1A1 in SJSA cells with low SULT1A1 levels. Overexpression of SULT1A1 resulted in the decrease of RNA pol II expression and less RNA synthesis upon RITA and its analogs treatment (Supplementary Fig. S4B and S4C). Interestingly, upon overexpression of SULT1A1 NSC782846 and NSC777196 induced less γH2AX compared with RITA (Supplementary Fig. S4B).

We also performed experiments on dose dependence of effects of RITA analogs in the WT cell line to further elucidate their MoA. Our data show that even a very low dose—0.25 μmol/L NSC777196 and 1 μmol/L NSC782846—can significantly downregulate oncogenes upon p53 activation (Fig. 5E). Notably, 1 μmol/L NSC782846 is sufficient to inhibit cell proliferation (Fig. 1C). As shown in Fig. 5F and Supplementary Fig. S4D, 1 μmol/L NSC782846 did not inhibit RNA Pol II nor block RNA synthesis, which indicate that the inhibition of oncogenes and growth suppression upon 1 μmol/L NSC782846 are indeed regulated by reactivated p53 but not due to global inhibition of transcription. In line with our results shown above, effects of NSC777196 on DNA damage and transcriptional block were weaker than that of RITA, but stronger than those induced by NSC782846 (Fig. 5).

Thus, we conclude that NSC777196 and NSC782846 are much less capable of induction of DNA damage and block of transcription, in line with previous study (14) showing that RITA analogs, especially NSC782846, have higher transition state barriers for the spontaneous formation of a reactive carbocation.

Combination of low-dose NSC782846 with 4-OH tamoxifen shows a synergetic effect in ER-positive breast cancer

On the basis of the tight relationship of the effect of our compounds and p53 as well as its negative regulators, MDM2 and MDM4, we used cBioPortal for Cancer Genomics to analyze the frequency of MDM2, MDM4, and TP53 gene alterations (Study of Origin; refs. 26, 27). MDM2 and MDM4 gene amplification frequency in breast cancer are 4% and 15%, respectively (Fig. 6A). As shown in Table 1, both MDM2/MDM4 amplifications and TP53 mutations have the tendency to be mutually exclusive.

Figure 6.

Synergistic effect of p53-targeting compound and ER-targeting 4-OH tamoxifen. A, Genetic alterations of MDM2, MDM4, and TP53 in breast cancer obtained from the cBioPortal from the Cancer Genomics database. B, Fraction of genome alteration stratified with ER status in the primary tumors (“+” indicates ER positive; “−” indicates ER negative). Each dot represents a patient. Red dots represent patients with MDM2 and MDM4 amplification (MDM2 ***, P < 0.001; MDM4 0.05<P < 0.1). C, MDM2 and MDM4 protein levels upon non-lethal dose treatment by NSC782846 in MCF7 WT cells as detected by Western blotting. D, Correlation of baseline MDM2 mRNA level with sensitivity to RITA, NSC777196, and NSC782846 in NCI-60 cell line database. E, Synergy between 4OH-TMX and low dose of NSC782846 in ER-positive MCF7 cells, as assessed using long-term colony formation assay. F, MCF7 cells were exposed to various concentrations of NSC782846 (0–4 μmol/L) and 4-OH TMX (0–8 μmol/L) for 48 hours and cell viability was assessed by cell prolifearion assay (left). The difference between single and combined treatments is statistically significant (**, P < 0.01; ***, P < 0.001). The Fa-CI plot shows the combined index value (CI) for each fractional effect (right). NSC782846 had a synergistic effect with 4-OH TMX (CI < 1).

Figure 6.

Synergistic effect of p53-targeting compound and ER-targeting 4-OH tamoxifen. A, Genetic alterations of MDM2, MDM4, and TP53 in breast cancer obtained from the cBioPortal from the Cancer Genomics database. B, Fraction of genome alteration stratified with ER status in the primary tumors (“+” indicates ER positive; “−” indicates ER negative). Each dot represents a patient. Red dots represent patients with MDM2 and MDM4 amplification (MDM2 ***, P < 0.001; MDM4 0.05<P < 0.1). C, MDM2 and MDM4 protein levels upon non-lethal dose treatment by NSC782846 in MCF7 WT cells as detected by Western blotting. D, Correlation of baseline MDM2 mRNA level with sensitivity to RITA, NSC777196, and NSC782846 in NCI-60 cell line database. E, Synergy between 4OH-TMX and low dose of NSC782846 in ER-positive MCF7 cells, as assessed using long-term colony formation assay. F, MCF7 cells were exposed to various concentrations of NSC782846 (0–4 μmol/L) and 4-OH TMX (0–8 μmol/L) for 48 hours and cell viability was assessed by cell prolifearion assay (left). The difference between single and combined treatments is statistically significant (**, P < 0.01; ***, P < 0.001). The Fa-CI plot shows the combined index value (CI) for each fractional effect (right). NSC782846 had a synergistic effect with 4-OH TMX (CI < 1).

Close modal
Table 1.

Correlations among alterations in TP53, MDM2, and MDM4 in breast cancer.

ABNeitherA not BB not ABothLog2 odds ratioPQTendency
MDM2 TP53 1,161 68 675 14 −1.498 <0.001 <0.001 Mutual exclusivity 
MDM4 TP53 1,177 52 674 15 −0.989 0.011 0.017 Mutual exclusivity 
MDM2 MDM4 1,772 79 64 0.072 0.554 0.554 Co-occurrence 
ABNeitherA not BB not ABothLog2 odds ratioPQTendency
MDM2 TP53 1,161 68 675 14 −1.498 <0.001 <0.001 Mutual exclusivity 
MDM4 TP53 1,177 52 674 15 −0.989 0.011 0.017 Mutual exclusivity 
MDM2 MDM4 1,772 79 64 0.072 0.554 0.554 Co-occurrence 

Note: Study of origin: Breast Cancer (MSK, Cancer Cell 2018).

We analyzed the relationship between the ER status of the primary breast cancer and MDM2/MDM4 amplification. Excluding 27 patients with unknown ER status, we found that in ER-positive patients MDM2 is amplified significantly more frequently (Fig. 6B). Although there was no statistical difference in MDM4 gene alterations between ER-positive and -negative patients (0.05 < P < 0.1), it might become more clear upon analysis of a larger patient cohort.

Our analysis suggests that ER-positive patients might benefit from compounds depleting MDM2/4. Low doses of compounds NSC782846 can activate WT p53 (Fig. 5E) and inhibit MDM2/MDM4 (Fig. 6C), similar to downregulation of MDM2 and MDM4 by RITA, as we previously published (28). In addition, the activity of RITA in NCI-60 cell lines is weakly associated with mRNA expression of p53 binding partner MDM2 (R2 = 0.22). However, the growth suppression by NSC777196 and NSC782846 is much more strongly associated with baseline MDM2 expression (R2 = 0.38 and 0.47, respectively; Fig. 6D), suggesting that interference with p53/MDM2 binding may be more important for NSC782846 activity.

Thus, we tested whether combination of 4-OH tamoxifen (4-OH TMX) with NSC782846 has an additive or synergistic effect on ER-positive breast cancer. Indeed, we observed much stronger growth suppression upon the 2 days pretreatment with 4OH-TMX combined with a low dose of NSC782846, as tested in ER-positive MCF7-WT cells (Fig. 6E). As shown in Fig. 6F, appropriate doses of 4OH-TMX slightly inhibited the viability of ER-positive breast cancer cells. However, a combination of NSC782846 and 4-OH TMX resulted in a much stronger antiproliferative effect compared with those of individual agents. NSC782846 had a synergistic effect with 4-OH TMX (CI < 1) when used in combination in MCF7 breast cancer cells.

In conclusion, p53-activating compounds with mechanism similar to NSC782846 might be good candidates for a personalized therapy of ER-positive patients with breast cancer in combination with 4-OH TMX–mediated endocrine therapy.

Small-molecule RITA has been previously identified by us using a phenotypic screen of the NCI compounds library. RITA displayed efficient induction of apoptosis by inhibiting MDM2 and MDM4 in cancer cells as well as in mouse xenografts (25, 28, 29). The exact mechanism of RITA action remains to be elucidated; in addition to p53-dependent growth suppression, it has strong p53-independent effects in cancer cells (16, 20, 30). Recently we have further elucidated the details of RITA interaction with p53 and revealed that RITA prevents the interaction between p53 and MDM2/MDM4 via allosteric mechanism (31).

p53-independent cytotoxic effects of RITA are dependent on phenol sulfotransferase SULT1A1, indicating that RITA is most likely a prodrug activated by sulfonation, with a mechanism dependent on the carbocation generation (32). The observed high toxicity of RITA in lungs of rats, dogs, and monkeys relative to mice (14) could be explained by the complete lack of phenol sulfotransferase activity in mouse lung cytosol (15). Thus, lung toxicity of RITA is the most important problem to be addressed. Our results showed that RITA analog NSC782846 can induce cancer cell apoptosis in the absence of SULT1A1. However, even if there was no DNA damage in the absence of SULT1A1 by NSC782846, another analog, NSC777196, still able to induce DNA damage and partially inhibit RNA pol II. Thus, the possibility remains that other sulfotransferases (such as SULT1A2, which is co-expressed with SULT1A1) could be responsible for activation of the NSC777196, whereas NSC782846 is much less prone to sulfonation. Previous in vivo experiments showed that NSC782846 caused tumor regression at the MTD without loss of weight (14). More specifically, lung toxicity detection with these RITA analogs in rats or other mammals needs to be explored in the future.

Another p53-independent effect of RITA is that it can inhibit RNA Pol I- and Pol II–mediated transcription by ROS-mediated DNA damage (20), thereby broadly repress transcription, including oncogene expression. In our study, we found that a low dose of NSC782846 activates p53, its target genes and represses oncogenes without inhibiting the transcription process and in the absence of DNA damage. Taken together, our data provide compelling evidence that RITA analog NSC782846 is indeed specifically targeting p53 in cancer cells. We also speculate that the off-target effect of RITA inducing p53 via DNA damage masks the direct effect of RITA on p53. Although the Pol II–mediated transcription repression upon high dose of NSC782846 can still be observed, it might be due to the low-efficiency production of carbocation metabolites by sulfate elimination from sulfonated products causing DNA damage in WT cell lines, as shown in our model in Supplementary Fig. S5.

The multi-gene nature of cancer suggests that the targeting of a single gene is unlikely to produce a sustained effect (33). Targeting two or more unique alterations in cancer cells appears to be a more feasible approach to achieve therapeutic efficiency and selectivity and to prevent the development of drug resistance. Importantly, upon combination treatment the clinical toxicities of each compound will not overlap to permit their use in effective doses (34–36). Our data suggest that it might be safe to use low doses of p53-specific RITA analogs, such as NSC782846, without SULT1A1-dependent lung toxicity, in combination with other target-specific drugs.

G. Selivanova reports grants from Swedish Cancer Society, Swedish Research Council, Knut and Alice Wallenberg foundation, and Eurostars during the conduct of the study; as well as reports a patent for 1-Azabicyclo[2.2.2]octan-3-one derivatives and maleimide derivatives and their use for treating tumors US692176, EP1476166; 2004-11-17 issued, a patent for Use of low-molecular weight compounds for preparing a medicament useful in treating mutant p53-mediated diseases EP1605939; 2008-10-15 issued, a patent for New Compounds and use thereof WO05/090341; 2011-11-15 issued, and 5. NOVEL THIOREDOXIN REDUCTASE INHIBITORS, EP21217702.6; 2021-12-24 pending; and is a co-founder of the small biotech company Aprea Therapeutics AB. No disclosures were reported by the other authors.

Y. Zhan: Conceptualization, formal analysis, investigation, writing–original draft. X. Zhou: Data curation, software, formal analysis. S. Peuget: Conceptualization, formal analysis, writing–review and editing. M. Singh: Methodology. B.D. Peyser: Software, formal analysis, writing–review and editing. Z. Fan: Project administration. G. Selivanova: Supervision, funding acquisition.

This work was supported by the grants to G. Selivanova from Swedish Research Council and Swedish Cancer Society. This work was supported in part by the Division of Cancer Treatment and Diagnosis of the National Cancer Institute, National Institutes of Health. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. We thank China Scholarship Council (CSC) supporting the research and work of Y. Zhan (No. 202006170200).

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.

Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/).

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Supplementary data