MCB-613 causes rapid and selective depletion of p53-R175H

We identified that a small-molecule compound called MCB-613 caused a rapid and sustained decrease in the level of the usually stable p53-R175H GOF mutant in the ovarian cancer cell line TYK-Nu (Fig. 1a, b and Supplementary Fig. 1A). Interestingly, in contrast to the effect on p53-R175H, a slight increase in the level of p53-WT protein was observed upon MCB-613 treatment in ALST cells (Fig. 1c). Furthermore, MCB-613 treatment had minimal effects on the other frequently observed GOF mutp53 (R248Q, R273H, and Y220C) in multiple cell lines (Fig. 1d,e and Supplementary Fig. 1B). To determine whether the effect of MCB-613 on p53-R175H mutant is specific to the ovarian cancer cell line TYK-Nu or mediated through a conserved mechanism, we tested the effect of MCB-613 on p53-R175H in TOV-112D (ovarian cancer) and SK-BR-3 (breast cancer) cells. Similar to the results using TYK-Nu cells, MCB-613 treatment resulted in dramatic decrease in p53-R175H levels in both TOV-112D and SK-BR-3 cells (Fig. 1f,g). Consistent results were also observed using ectopically expressed p53-R175H, p53-R273H, and p53-WT in the p53-null SKOV3 cells upon MCB-613 treatment, further suggesting that the effect of the small molecule MCB-613 on the conformational p53-R175H mutant is mediated through a conserved mechanism (Fig. 1h).

Fig. 1 MCB-613 causes a rapid and selective decrease in the level of p53-R175H. a MCB-613 (6 µM) caused a rapid decrease in p53-R175H protein levels in TYK-Nu cells. b Effect of different concentrations of MCB-613 (2 h) on p53-R175H levels in TYK-Nu cells. MCB-613 treatment resulted in the decrease in p53-R175H levels in a concentration dependent manner. c p53-WT protein levels in ALST cells slightly increased upon MCB-613 (6 µM, 2 h) treatment. d, e MCB-613 (2 h) had minimal effect on other GOF mutp53, such as d p53-R248Q levels in OVCAR3 and S1GODL cells, and e p53-R273H levels in OVCA420 and MDAH2774 cells. f, g p53-R175H levels decreased in multiple cell lines upon MCB-613 (2 h) treatment; f TOV-112D (ovarian cancer); g SK-BR-3 (breast cancer). g Effect of MCB-613 (2 h) on GFP-tagged p53-WT, and p53-R175H mutant protein in the p53−/− SKOV3 cells. h Effect of MCB-613 (2 h) on GFP-tagged p53-WT, p53-R175H, and p53-R273H mutants in SKOV3 cells. i Immunofluorescence shows decrease in p53-R175H levels (green) in TYK-Nu cells upon MCB-613 treatment (2 h). j Effect of MCB-613 (2 h) on GFP-tagged p53 GOF mutants in SKOV3 cells Full size image

Because the p53-R175H mutant is known to form aggregates in cells, we sought to confirm if MCB-613 caused reduction of p53-aggregates. Both immunofluorescence and native gel-western blot analyses revealed that MCB-613 treatment indeed resulted in a dramatic decrease in total p53-R175H protein levels, including aggregates (Fig. 1i and Supplementary Fig. 1C). To determine the effect of MCB-613 on other mutp53 proteins frequently observed in human cancers, and to ascertain whether the effect was specific to p53-R175H, we expressed frequently observed p53 mutants in SKOV3 cells, and evaluated the effect of MCB-613 on their levels. The effect of MCB-613 was found to be most profound on p53-R175H mutant protein. In addition to p53-R175H, the levels of C176Y, H179R, and H193R mutants also decreased slightly upon MCB-613 treatment (Fig. 1j). It is possible that mutations in the region near the R175 codon could potentially induce structural changes that are similar to those induced by R175H, and is important in mediating the response to MCB-613. Further, no noticeable effect was observed in the levels of the Y220C, S241F, R248Q, and C277H mutants upon MCB-613 treatment (Fig. 1j).

Effect of MCB-613 on p53-R175H is independent of steroid receptor co-activators

MCB-613 is a known small-molecule stimulator of the steroid receptor co-activators (SRCs), and also causes rapid turnover of SRC-1, SRC-2, and SRC-3 proteins in cells (Supplementary Fig. 2A)21. To ascertain if the effect of MCB-613 on p53-R175H is mediated through an SRC dependent transcriptional mechanism, we measured the changes in p53 transcript levels in ALST and TYK-Nu cells, upon MCB-613 treatment. The absence of correlation between the p53 mRNA and protein levels, upon MCB-613 treatment in both ALST and TYK-Nu cells, confirmed that the effect of MCB-613 on p53-WT and p53-R175H levels was independent of direct SRC dependent transcriptional regulation (Fig. 2a). MCB-613 also had no significant effect on the p53 mRNA levels in OVCAR3 (R248Q) and OVCA420 (R273H) cells (Supplementary Fig. 2B). To ascertain whether the effect on p53-R175H is dependent on SRCs, we used siRNA to knockdown SRC-1, SRC-2, or SRC-3 in TYK-Nu and ALST cells. The knockdown of SRC-1, SRC-2, or SRC-3 had no effect on either p53-WT or p53-R175H mutant protein levels (Fig. 2b–e). The SRC-1 levels, in ALST cells, were very low and undetectable by western blot analysis (Fig. 2b). Further, the small-molecule SRC-3 inhibitor, SI2, did not alter p53-R175H levels (Fig. 2f). Collectively, these results confirmed that the effect of MCB-613 on p53-WT and the p53-R175H mutant is independent of the transcriptional co-activators SRC-1, SRC-2, and SRC-3 in these cells.

Fig. 2 MCB-613 causes lysosome-mediated degradation on p53-R175H. a Effect of MCB-613 (2 h) on p53 mRNA levels in ALST and TYK-Nu cells. Values are normalized mean ± s.e.m. (n = 3). b–d siRNA-mediated knockdown (48 h) of SRC-1 (b), SRC-2 (c), and SRC-3 (d) had no effect on p53-WT (ALST cells) and p53-R175H (TYK-Nu cells) levels. e shRNA-mediated knockdown of SRC3 (72 h) in TYK-Nu cells does not affect p53-R175H levels. f SRC-3 inhibitor SI2 (50 nM, 2 h) has no effect on p53-R175H levels in TYK-Nu cells. g, h MCB-613 caused a g decrease in the half-life of p53-R175H and h increase in the half-life of p53-WT. i Proteasome inhibitor MG132 does not inhibit MCB-613 induced turnover of p53-R175H. j Lysosome inhibitors (pepstatin A, Leupeptin, and E-64D) rescued MCB-613 induced turnover of p53-R175H Full size image

MCB-613 causes lysosome-mediated turnover of p53-R175H

To determine whether p53-WT and the R175H mutant are regulated by MCB-613 at the translational level or through a post-translational mechanism, we treated ALST and TYK-Nu cells concurrently with MCB-613 and the protein synthesis inhibitor cycloheximide. Cycloheximide-chase analysis revealed that, MCB-613 causes a dramatic decrease in the half-life of the usually stable p53-R175H mutant protein (Fig. 2g). In contrast to the effect on R175H mutant, the half-life of p53-WT protein increased in the presence of MCB-613, resulting in its accumulation, indicating the effect of MCB-613 on p53-WT and R175H mutant to be post-translational (Fig. 2h). In comparison, MCB-613 had no effect on the turnover of p53-R248Q and p53-R273H proteins at these early-time points (Supplementary Fig. 2C, D). Because MCB-613 causes increased p53-R175H turnover, we sought to determine whether the effect is mediated via the proteasome or the lysosomal pathway. The proteasome inhibitor MG132 was unable to rescue the MCB-613 induced turnover of the p53-R175H mutant protein, suggesting that the protein is not targeted to the proteasome for degradation (Fig. 2i). In contrast, treatment of TYK-Nu cells with lysosomal inhibitors reversed the effect of MCB-613, thereby demonstrating that the drug causes selective turnover of the p53-R175H GOF mutant by targeting it to the lysosome (Fig. 2j). The autophagy inhibitor, LY294002, also reversed the effect of MCB-613 on p53-R175H, suggesting a potential role for autophagosomes in MCB-613-induced p53-R175H turnover (Supplementary Fig. 2E). Interestingly, MCB-613 was previously reported to induce the formation of autophagosomes in breast cancer cells21.

Interestingly, MCB-613 causes rapid export of the p53-R175H mutant protein from the nucleus to the cytoplasm (Fig. 3a and Supplementary Fig. 3A). No change in p53 localization was observed in cells expressing p53-WT or the R248Q and R273H mutants (Fig. 3b–d and Supplementary Fig. 3B). Further, consistent with being targeted to the lysosome for degradation, p53-R175H localized to the lysosome upon MCB-613 treatment (Fig. 3e). We also observed co-localization of p53-R175H with calnexin, an endoplasmic reticulum (ER) marker (Fig. 3f). Although the significance of ER localization is not entirely clear, we hypothesize that localization to ER might serve as an intermediate step in targeting the R175H mutant protein to the lysosome for degradation, in response to MCB-613. No significant localization of the R175H mutant was observed with Mitotracker, a mitochondria-specific dye (Supplementary Fig. 3C).

Fig. 3 MCB-613 causes nuclear export of p53-R175H protein. a–d MCB-613 caused a rapid export of p53-R175H (TYK-Nu cells) protein but not b p53-WT (ALST cells) or other GOF mutp53 c p53-R273H (OVCA420 cells), and d p53-R248Q (OVCAR3 cells). e, f Upon MCB-613 treatment, p53-R175H (green) co-localizes with e lysosomes (red; lysotracker) and f endoplasmic reticulum (red; α-calnexin) in TYK-Nu cells Full size image

MCB-613 treatment causes rapid ubiquitination of p53-R175H

A short MCB-613 treatment (<20 min) results in the accumulation of higher migrating p53 isoforms in TYK-Nu cells (R175H), but not in ALST (WT) or OVCA420 (R273H) cells as shown by western blot analyses (Fig. 4a). To ascertain if these higher molecular-weight bands represent ubiquitinated forms of the R175H mutant, we immunoprecipitated mutp53 protein from the control and MCB-613 treated TYK-Nu cells, and performed an anti-ubiquitin western blot. A marked increase in ubiquitinated p53-R175H protein was observed upon MCB-613 treatment (Fig. 4b). To confirm this result, we ectopically expressed His-tagged ubiquitin (His-Ub) in TYK-Nu cells, and performed Ni-NTA pull down of cellular proteins containing His-Ub from the control and MCB-613 treated cells, followed by an anti-p53 immunoblot. Consistent with our previous result, MCB-613 treatment results in increased pull down of ubiquitinated p53-R175H protein (Supplementary Fig. 4A). Increased ubiquitination of p53-R175H mutant protein, in response to MCB-613 treatment, is also observed in SKOV3 cells expressing ectopic p53-R175H and His-Ub, suggesting the mechanism to be conserved across different cell types (Fig. 4c). The levels of both K-48 and K-63-linked ubiquitin chains are enhanced on p53-R175H, in response to MCB-613 treatment (Supplementary Fig. 4B and C). However, the increase in K-63-linked ubiquitin chain appeared to be more dominant, consistent with lysosomal export of the ubiquitinated protein (Supplementary Fig. 4C). To identify the regions within p53-R175H protein, important in mediating sensitivity to MCB-613, we expressed N and C-terminal truncated versions of p53-R175H in SKOV3 cells. Deletion of the N-terminal 42 amino acids (43–393) or C-terminal 30 amino acids (1–363) made p53-R175H resistant to MCB-613-induced degradation (Supplementary Fig. 4D). WT p53 has 20 lysine residues that can be potentially ubiquitinated (Supplementary Fig. 4E). To determine if any of the lysines within the N-terminal or C-terminal region, identified to mediate sensitivity to MCB-613, is critical for the response, we used site-directed mutagenesis to generate lysine-to-arginine mutants and expressed them in SKOV3 cells. None of the single or double lysine substitution mutants we tested, resulted in resistance to MCB-613 induced turnover of the R175H mutant (Supplementary Fig. 4F). The inability of the tested lysine substitution mutants to resist MCB-613 induced degradation suggests: (i) possible redundancy in ubiquitination sites on p53-R175H or (ii) that the C-terminal and N-terminal regions do not harbor the critical ubiquitination sites, but are important for mediating protein–protein interactions necessary for MCB-613 induced ubiquitination and turnover of the R175H mutant.

Fig. 4 MCB-613 causes increased ubiquitination of p53-R175H protein. a Higher migrating p53-R175H bands accumulate in TYK-Nu cells upon short-term (<20 min) MCB-613 treatment; but not in ALST (p53-WT) or OVCA420 (p53-R273H) cells. b MCB-613 caused increased ubiquitination of p53-R175H in TYK-Nu cells. c Increased ubiquitination of ectopic p53-R175H-HA upon MCB-613 treatment in SKOV3 cells. d Effect of increasing concentrations of H 2 O 2 (1 h) on p53-R175H levels in TYK-Nu cells. e Effect of increasing concentrations of H 2 O 2 (1 h) on p53-WT levels in ALST cells. f, g MCB-613 causes decrease in MDM2 levels in f TYK-Nu and g SKBR-3 cells. h Effect of MCB-613 on MDM2 mRNA levels in ALST and TYK-Nu cells. Values are normalized mean ± s.e.m. (n = 3). i MDM2 knockdown (48 h) has no effect on p53-R175H levels in TYK-Nu cells. j Effect of MCB-613 on p53-R175H turnover is independent of MDM2. k MG132 reversed MCB-613 induced MDM2 turnover in ALST cells Full size image

MDM2 mediates the effect of MCB-613 on p53-WT

Previous studies have shown that MCB-613 causes oxidative stress in cells21. To determine whether MCB-613-induced oxidative stress mediates the effect on p53-WT and R175H mutant, we treated TYK-Nu and ALST cells with increasing concentrations of H 2 O 2 for 1 h. While p53-WT levels decreased slightly at higher H 2 O 2 concentrations, H 2 O 2 has no effect on p53-R175H levels (Fig. 4d, e). These results indicate that the effect of MCB-613 on p53-WT and R175H mutant is not mediated by cellular oxidative stress. WT-p53 levels in cells are tightly regulated by the ubiquitin-proteasome system. The E3 ligase MDM2 is known to ubiquitinate p53-WT and target it for degradation. Although the role of MDM2 in p53-WT degradation is well-established, its role in regulating the turnover of the p53-R175H mutant is not clear22. Recent reports have identified several small molecules that can bind mutp53 and induce conformational changes that make the p53 mutants assume a WT-like conformation (a process termed “reactivation”), and thereby become a target of MDM2-mediated turnover1. To determine if MCB-613 operates through such a mechanism, we tested the ability of nutlin-3A to reverse the effect of MCB-613 on p53-R175H in TYK-Nu cells. Nutlin-3A has been shown to stabilize p53-WT by disrupting p53-MDM2 interaction23. Nutlin-3A did not reverse the effect of MCB-613 on p53-R175H, thereby confirming that MCB-613 does not cause reactivation of p53-R175H (Supplementary Fig. 4G). Consistently, we found that MCB-613 does not bind to recombinant p53-R175H protein (Supplementary Fig. 4H). Interestingly, MCB-613 causes a rapid decrease in MDM2 levels in multiple cell lines (Fig. 4f, g, k). The effect of MCB-613 on MDM2 was not transcriptional (Fig. 4h). MDM2 mRNA slightly increased in ALST cells upon MCB-613 treatment, possibly due to the transcriptional feedback from elevated p53-WT protein in these cells (Fig. 4h). No change in MDM2 mRNA was observed upon MCB-613 treatment in TYK-Nu cells (Fig. 4h). The decrease in MDM2 protein levels would explain the stabilization of p53-WT in ALST cells, but we wanted to determine if MDM2 mediated the decrease in p53-R175H stability. siRNA knockdown of MDM2 had no effect on the p53-R175H levels (Fig. 4i). Further, MCB-613 caused a dramatic reduction in p53-R175H levels even in the absence of MDM2 in both TYK-Nu cells and p53−/−:MDM2−/− MEFs, expressing ectopic p53-R175H (Fig. 4j and Supplementary Fig. 4I). Collectively, these results demonstrate that MDM2 mediates the effect of MCB-613 on p53-WT, but the effect on p53-R175H is independent of MDM2. Moreover, MCB-613 caused increased ubiquitination of MDM2 (Supplementary Fig. 4J). To determine whether MDM2 is targeted to the proteasome for degradation upon MCB-613 treatment, we treated ALST cells simultaneously with both MCB-613 and proteasome inhibitor MG132. Concurrent MG132 treatment rescued MCB-613 induced MDM2 degradation, suggesting that MCB-613 causes proteasome-mediated turnover of MDM2 in ALST cells (Fig. 4k).

DUB inhibitors mimic the effect of MCB-613 on p53-R175H

Since MCB-613 treatment resulted in increased ubiquitination of p53-R175H, we hypothesized that MCB-613 might be acting through pathways that either augment the activity of specific ubiquitin ligases, or inhibit the activity of DUBs. Connectivity map (CMap) analysis of the RNAseq data, from MCF-7 cells treated with MCB-613 with the publically available RNAseq data post-treatment with other chemical compounds, revealed remarkable functional similarity between MCB-613 and a structurally similar DUB inhibitor, NSC632839 (Fig. 5a–c). We tested the ability of NSC632839 to mimic the effect of MCB-613 on p53-R175H and p53-WT. Similar to MCB-613, NSC632839 treatment results in the depletion of p53-R175H and a slight increase in p53-WT (Fig. 5d,e). NSC632839 treatment also causes a decrease in MDM2 levels (Fig. 5d). Further, the reversible cell permeable pan-DUB inhibitor PR-619 also causes a decrease in p53-R175H and MDM2 levels in TYK-Nu cells (Fig. 5f). Consistent with previous results using MCB-613 and NSC632839, an increase in p53-WT was observed upon PR-619 treatment (Fig. 5g). Similar to MCB-613, the effect of PR-619 was also conserved in SKOV3 cells expressing ectopic p53-R175 H (Fig. 5h). Again, similar to MCB-613, PR-619 causes export of nuclear p53-R175H protein into the cytoplasm (Fig. 5i). In contrast to its effect on p53-R175H and p53-WT, PR-619 and NSC632839 did not alter the levels of p53-R273H mutant in either OVCA420 cells or SKOV3 cells expressing GFP-tagged p53-R273H (Fig. 5j,k), suggesting the effect is selective to p53-R175H. Unlike PR-619 and NSC632839, the USP14 inhibitor IU-1 did not alter the levels of either p53-WT or the R175H mutant, suggesting specificity in DUBs involved in mediating the stability and turnover of p53-WT and the R175H mutant (Supplementary Fig. 5A).

Fig. 5 DUB inhibitors mimic the effect of MCB-613 on p53-WT and p53-R175H. a Comparison of RNAseq data from MCF-7 cells treated with MCB-613 (6 h) and the DUB inhibitor NSC632839. b Venn diagram summarizing the result in Fig. 5a shows that the gene-expression changes induced by MCB-613 (6 h) and the DUB inhibitor NSC632839 in MCF-7 cells are highly similar. c Chemical structure of MCB-613 and NSC632839. d, e NSC632839 (20 µM, 2 h) causes d depletion of p53-R175H and MDM2 and e slight increase in p53-WT levels. f, g PR-619 (10 µM, 2 h) causes f depletion of p53-R175H and MDM2 and g slight increase in p53-WT levels. h Effect of MCB-613 (6 µM, 2 h) and the pan-DUB inhibitor PR-619 (10 µM, 2 h) on ectopically expressed GFP tagged p53-R175H protein and endogenous MDM2 in SKOV3 cells (p53-null). i PR-619 causes cytoplasmic localization of p53-R175H (green). j Effect of PR-619 (10 µM) on p53-R273H mutant protein in OVCA420 cells. k Effect of DUB inhibitors PR-619 (10 µM, 2 h) and NSC632839 (20 µM, 2 h) on ectopically expressed GFP-tagged p53-R273H in the p53-null SKOV3 cells Full size image

USP15 regulates the stability of p53-R175H GOF mutant

Because the DUB inhibitor NSC632839 successfully mimicked the response elicited by MCB-613 treatment on p53-WT and the R175H mutant, we decided to test the role of USP7, a known NSC632839 targeted DUB24, on the stability and turnover of p53-WT and p53-R175H mutant. siRNA-mediated knockdown of USP7, in ALST cells, resulted in elevated p53-WT levels similar to those observed with MCB-613 and DUB inhibitors (Fig. 6a). This result is consistent with the previously known role of USP7 in regulating p53-WT stability25. However, USP7 knockdown in TYK-Nu cells did not result in any noticeable reduction in p53-R175H levels (Fig. 6b). This suggested that the effect of DUB inhibitors on p53-WT and p53-R175H is mediated through potentially different DUBs. USP15 was recently described as capable of reducing MDM2 levels in cells26. Since PR-619 is known to inhibit USP1527 (Supplementary Fig. 6C), we tested the effect of USP15 knockdown on p53-WT and p53-R175H levels. In contrast to previously reported effects on p53-WT26, siRNA-mediated knockdown of USP15 did not cause any change in p53-WT levels in ALST cells (Fig. 6c). Interestingly, knockdown of USP15 in both TYK-Nu and TOV112D cells caused p53-R175H levels to decrease (Fig. 6d and Supplementary Fig. 6A). USP15 knockdown did not alter the levels of p53-R273H in OVCA420 cells or p53-R248Q in OVCAR3 cells (Fig. 6e, f). These results show that WT and the different mutp53 proteins are regulated differentially by DUBs in cancer cells, and establish USP15 as an upstream regulator of p53-R175H in ovarian cancer cells. Next, we performed p53-immunoprecipitation, from the p53-R175H mutant expressing TYK-Nu cells, to determine whether p53-R175H interacted with USP15. The p53-null SKOV3 cells served as a control for non-specific antibody interactions. USP15 was found to co-immunoprecipitation (co-IP) with p53-R175H in TYK-Nu cells (Fig. 6g). No interaction was observed by co-IP between USP15 and p53-WT or other GOF mutants (p53-R248Q and p53-R273H) (Supplementary Fig. 6B). Since MCB-613 treatment induced increased ubiquitination of p53-R175H, we tested the ability of USP15 to mimic this response. p53-R175H was immunoprecipitated from TYK-Nu cells treated with either non-targeting siRNA or USP15 siRNA. Anti-ubiquitin immunoblotting of p53-IP revealed increased ubiquitination of p53-R175H upon USP15 knockdown (Fig. 6h). In contrast, USP15 knockdown did not mediate increased ubiquitination of p53-R273H in OVCA420 cells (Fig. 6i). Further, the depletion of p53-R175H upon USP15 knockdown was rescued by co-treatment with lysosomal inhibitors (pepstatin-A, leupeptin, and E-64D), suggesting that similar to MCB-613, USP15 causes selective depletion of p53-R175H through an ubiquitin-mediated lysosomal pathway (Fig. 6j).

Fig. 6 USP15 regulates p53-R175H levels in ovarian cancer cells. a, b Effect of USP7 knockdown on a p53-WT and b p53-R175H levels. c, d USP15 knockdown causes c no effect on p53-WT levels (ALST cells) but d decrease in p53-R175H levels (TYK-Nu and TOV-112D cells). e, f siRNA-mediated knockdown of USP15 has no effect on the levels of e p53-R273H in OVCA420 cells and f p53-R248Q in OVCAR3 cells. g USP15 co-immunoprecipitates with p53-R175H. h Increased ubiquitination of p53-R175H upon USP15 knockdown. p53-R175H was immunoprecipitated from TYK-Nu cells treated with non-targeting siRNA and USP15 siRNA, using anti-p53 antibody (FL-393). Immunoblot for ubiquitin (anti-Ub (P4D1)) was performed on the immunoprecipitated proteins. i USP15 knockdown by siRNA does not result in increased ubiquitination of p53-R273H in OVCA420 cells. j Lysosomal inhibitors reversed the effect of USP15 siRNA on p53-R175H levels. k MCB-613 treatment (6 µM, 2 h) results in decrease in USP15 protein levels Full size image

To determine how MCB-613 might be interfering with USP15 function in cells, we first tested the possibility that MCB-613 functions as a direct inhibitor of USP15. However, unlike PR-619, MCB-613 did not cause a decrease in USP15 activity using the artificial substrate Ub-AMC in the in vitro DUB activity assay (Supplementary Fig. 6C and D). Further, no change in USP15 mRNA levels was observed upon MCB-613 treatment (Supplementary Fig. 6E). Interestingly, MCB-613 treatment resulted in decreased USP15 protein levels in TYK-Nu (R175H) and OVCA420 (R273H) cells, suggesting that MCB-613 causes depletion of USP15 protein through a post-translational mechanism (Fig. 6k and Supplementary Fig. 6F). These results are consistent with our previous observations that the effect of MCB-613 on p53-R175H is post-translational, and that depletion of USP15 causes decreases in p53-R175H levels in TYK-Nu cells, but not p53-R273H in OVCA420 cells.

USP15 knockdown reduced viability of p53-R175H cancer cells

GOF mutp53 proteins play important roles in the growth and survival of cancer cells, and selective depletion of mutp53 has been suggested as a potential strategy to induce targeted killing of cancer cells1. As expected, knockdown of mutp53 in ovarian cancer cells resulted in decreased cell viability (Fig. 7a and Supplementary Fig. 7A)28. Since MCB-613 causes selective depletion of p53-R175H mutant protein, we determined the sensitivity of ovarian cancer cell lines, expressing the different mutp53 to MCB-613. Both TYK-Nu and TOV-112D cells express the p53-R175H mutant, and were found to be more sensitive to MCB-613 treatment compared with ALST (p53-WT), OVCA420 (p53-R273H), and COV362 (p53-Y220C) cells (Fig. 7b). Increased sensitivity of TYK-Nu cells to cell death was also observed with the DUB inhibitor PR-619 (Fig. 7c).

Fig. 7 USP15 depletion causes cancer cell death in ovarian cancer cells expressing p53-R175H. a p53 knockdown causes decreased viability in ovarian cancer cells expressing GOF mutp53, but not p53-WT and p53-null cells. Values are normalized mean ± s.e.m. (n = 3; *p-value < 0.05). b Ovarian cancer cells expressing p53-R175H (TYK-Nu and TOV-112D) are more sensitive to MCB-613 than ALST (p53-WT), OVCA420 (p53-R273H), and COV362 (p53-Y220C). Values are normalized mean ± s.e.m. (n = 3). c TYK-Nu cells are more sensitive to PR-619 compared with ALST and OVCA420 cells. Values are normalized mean ± s.e.m. (n = 3; *p-value < 0.05). d Effect of USP15 knockdown on cell viability in ovarian cancer cells carrying different p53 mutation status. Values are normalized mean ± s.e.m. (n = 3; *p-value = 0.0164). e USP15 knockdown significantly reduced the anchorage-independent growth of TYK-Nu cells (p53-R175H). Values are normalized mean ± s.d. (n = 4; **p-value = 0.006). f Data retrieved from Oncomine showing elevated USP15 mRNA levels in multiple cancers from previously published datasets29, 39,40,41,42. g Schematic showing that different pathways regulate the stability of p53-R175H and p53-WT in ovarian cancer cells. MCB-613 and DUB inhibitors, such as NSC632839 and PR-619 caused selective depletion of the GOF p53 mutant p53-R175H, while causing slight increase in p53-WT levels. While inhibition of USP15 by these small molecules or siRNA resulted in increased ubiquitination and lysosome-mediated turnover of p53-R175H, it had no effect on p53-WT levels. In contrast, USP7 depletion caused MDM2-mediated stabilization of p53-WT protein Full size image

Further, USP15 knockdown resulted in decreased cell viability in TYK-Nu cells, but not in ALST, OVCAR3, or OVCA420 cells (Fig. 7d). In contrast, USP7 knockdown did not have any effect of TYK-Nu cell viability (Supplementary Fig. 7B). Colony forming assays revealed a significant decrease in the ability of TYK-Nu cells to form colonies upon USP15 knockdown, and colonies that did form were small (Fig. 7e). The USP15 gene is amplified in several cancers including ovarian, glioblastoma, and breast cancer29. Publically available data accessible through Oncomine show that USP15 expression is significantly elevated in ovarian serous cystadenoacrcinoma, lobular breast carcinomas, prostate cancer, cervical squamous cell carcinomas, and glioblastomas, suggesting potential clinical relevance for this protein are these cancers, especially in relation to the p53 status in these tumors (Fig. 7f and Supplementary Fig. 7C)29. Thus, taken together, this study implicates USP15 as a previously unknown clinically important regulator of p53-R175H mutant protein in ovarian cancer cells, and further illustrates that distinct pathways regulate the levels and stability of the different mutp53 proteins and WT-p53 in cells (Fig. 7g).