SMG1i and MDM2-p53 inhibitors exhibit synergistic inhibition of cell proliferation in HeLa cells
XR-2 is a novel small-molecule MDM2-p53 inhibitor synthesized in our laboratory. Similar to RG7388, XR-2 can activate p53 in MDM2-overexpressing TP53 wild-type tumor cells, thereby promoting cell apoptosis. As shown in Fig. 1a, XR-2 activated the expression of p53 in the TP53 wild-type HCT116 cells in a dose-dependent manner. HeLa cells express wild-type TP53 gene; however, the majority of translated p53 protein is degraded by the E6-E6AP-p53 complex(Hengstermann et al., 2001). To investigate the effect of XR-2 on p53 in HeLa cells, we treated HeLa cells with different concentrations of XR-2 for 24 hours. Treatments with up to 10 μM of XR-2 did not show any significant increase in the low basal p53 expression (Fig. 1b). Cheruiyot et al.(Cheruiyot et al., 2021) reported a novel SMG1 inhibitor, SMG1i, with high selectivity for SMG1 and effective inhibition of NMD. In this study, we used this agent to attenuate NMD activity. Previous studies by Gudikote et al. (Gudikote, et al., 2021) and our results shown in Fig. 1a indicated that inhibition of NMD could activate p53β/γ, two p53 isoforms generated from alternative splicing of full-length p53, in HCT116 cells. We found that SMG1i stimulated p53β/γ expression in HeLa cells as well (Fig. 1c). Furthermore, treatment with 3.3 µM or 10 µM of SMG1i for 24 hours induced apoptosis in HeLa cells, which was accompanied by significantly increased expression of the apoptosis markers cleaved-caspase-3 and cleaved-PARP (Fig. 1c). Our unpublished data showed that a co-treatment with XR-2 and SMG1i exhibits significant synergistic effects in inducing cell apoptosis and inhibiting cell cycle progression in TP53 wild-type tumor cells. To determine the combined effects on HeLa cells, we treated HeLa cells with different concentrations of these two agents alone or in combination. Interestingly, as shown in Fig. 1d and Fig. 1e, although HeLa cells were insensitive to XR-2 and SMG1i, the combination of these two agents significantly inhibited cell proliferation. To further assess the magnitude of the synergistic effect, we calculated the synergy score using the Bliss independence (Fig. 1f) and the Loewe additivity models (Supplementary Fig. 1), which yielded synergy scores of 19.504 and 18.629, respectively. The synergy score in both models was greater than 10, indicating significant synergistic interactions between the two agents. These two models also showed the most significant synergistic area between 0.2–3.2 µM of these two agents.
Combination of NMD inhibition and MDM2-p53 inhibitors synergistically induce apoptosis in HeLa cells
Next, we evaluated the effect of the combination of these two agents on apoptosis using western blotting. The results revealed that XR-2 or SMG1i monotherapy had a small effect on the apoptosis of HeLa cells at 3.3 µM (Fig. 2a). However, the combination treatment significantly increased the expression of apoptosis markers, cleaved-caspase-3 and cleaved-PARP and dramatically increased the DNA damage marker, γH2AX. These results suggested that combining these two agents results in genomic DNA instability and apoptosis. Another MDM2-p53 inhibitor, RG7388, also showed similar synergistic effects in inducing apoptosis in HeLa cells when combined with SMG1i (Fig. 2b), as evidenced by an increased expression of cleaved PARP. Furthermore, NMD inhibition via siRNA targeting UPF1 (Fig. 2c and Supplementary Fig. 2) exhibited significant synergistic effects when combined with the XR-2 treatment. These results indicated that the synergism observed between XR-2 and SMG1i is specific to the inhibition of MDM2-p53 and NMD, results in increased DNA damage, and induces cell apoptosis in HeLa cells.
Subsequently, we assessed apoptosis using flow cytometry. As shown in Fig. 2d and Fig. 2e, low concentrations (1 µM) of XR-2 or SMG1i had no effect on apoptosis, and higher concentrations (3.3 µM) of monotherapy slightly increased apoptosis in HeLa cells. However, combining of these two agents at 1 µM and 3.3 µM induced 20% and 50% of apoptotic HeLa cells, respectively, which indicated a dramatically synergistic effect of these two agents.
XR-2 and SMG1i co-treatment activates p53 pathway in HeLa cells
Next, we performed cell cycle analysis on HeLa cells upon treatment with XR-2, SMG1i, or a combination of these two agents by flow cytometry. As depicted in Fig. 3a and Fig. 3b, XR-2 and SMG1i treatments alone did not show any effect on cell cycle with no significant changes in the proportion of cells in G1- and S-phases. However, the combined treatment of the two agents resulted in significant changes in the cell cycle distribution with the majority of the cells confined to the G1-phase, which indicated that the combination treatment arrested the cell cycle and inhibited cell proliferation. Collectively, these results showed that the combination of these two agents results in growth arrest and increased apoptosis. Hence, we hypothesized that the combination treatment activates the p53 pathway. Using quantitative real-time PCR (Fig. 3c), we studied changes in the expression of four well-known p53 target genes, P21 and GADD45A, which are associated with cycle progression, and BAX and PUMA, which are linked to apoptosis. The treatment with XR-2 did not show any changes in the expression of these genes, whereas that with SMG1i showed a small increase in their expression. Consistent with increased apoptosis and cell cycle arrest, the combination treatment showed significant upregulation in the expression of these four genes, which was also accompanied by an increase in the TP53 expression.
MDM2-p53 inhibitor and NMD inhibitor act synergistically through truncated E6 protein
Taken together, these experiments suggested that the combined treatment with the MDM2-p53 and NMD inhibitors activated the p53 pathway. p53 is mainly degraded by the E6-E6AP-p53 complex in HeLa cells; thus, we hypothesized that the synergistic effects might be related to E6 expressed by the HPV genome. As shown in Fig. 4a, the E6 gene in HeLa cells was expressed in two forms. The full-length transcript (E6-FL) produced a protein with 158 amino acid residues. The other one underwent alternative splicing (E6*) and deficit of a 182 bp fragment, which resulted in the generation of a premature termination codon and yielded a protein of only 57 amino acid residues(Inagaki et al., 1988, Olmedo-Nieva et al., 2018). We designed a set of primers, as illustrated in Fig. 4a, to detect these isoforms. HeLa cells expressed the two isoforms simultaneously (Fig. 4b and Supplementary Fig. 3a), and the expression level of E6* was much higher than that of E6-FL. Subsequently, we constructed an E6*-overexpression plasmid using the PCDH backbone vector. E6* was overexpressed in HeLa cells, and the resulting cells were treated with these two agents alone or in combination. The results as shown in Fig. 4c and Supplementary Fig. 3b depicted that overexpression of E6* could rescue apoptosis caused by the combination treatment, indicating that the E6* protein is indeed involved in the observed synergistic effect.
We further performed RNA-Seq analysis on HeLa cells treated with XR-2, SMG1i, and the combination of these two drugs. As shown in Fig. 4d, monotherapy with XR-2 had no effect on the transcriptome, whereas SMG1i single treatment resulted in more than 2,000 of DEGs, and the number of DEGs was even higher in the combination treatment group. To identify the enriched signaling pathways, we chose the top 200 upregulated genes in the combination group and performed enrichment analysis using the MSigDB Hallmark database (Fig. 4e). TNF-α and p53 were the most enriched pathways, apoptosis, unfolded protein response, and hypoxia were also significantly enriched. In summary, the combined treatment activated the p53 pathway along with an increased unfolded protein cellular stress response and hypoxia, eventually leading to cell apoptosis.