Targeted MDM2 degradation reveals a new vulnerability for p53 inactivated triple negative breast cancer

Abstract

Triple negative breast cancers (TNBC) frequently inactivate p53, increasing their aggressiveness and therapy resistance. We identified an unexpected protein vulnerability in p53-inactivated TNBC and designed a new PROTAC to target it. Our PROTAC selectively targets MDM2 for proteasome-mediated degradation with high-affinity binding and VHL recruitment. MDM2 loss in p53 mutant/deleted TNBC cells in 2D/3D culture and TNBC patient explants, including relapsed tumors, causes apoptosis, while sparing normal cells. Our MDM2-PROTAC is stable in vivo, and treatment of TNBC xenograft-bearing mice demonstrates tumor on-target efficacy with no toxicity to normal cells, significantly extending survival. Transcriptomic analyses revealed upregulation of p53 family target genes. Investigations showed activation and a required role for TAp73 to mediate MDM2-PROTAC-induced apoptosis. Our data, challenging the current MDM2/p53 paradigm, show MDM2 is required for p53-inactivated TNBC cell survival, and PROTAC-targeted MDM2 degradation is an innovative potential therapeutic strategy for TNBC and superior to existing MDM2 inhibitors.

INTRODUCTION

Cancers that inactivate the p53 tumor suppressor, through mutation or deletion, are more aggressive and have increased resistance to many therapies (1,2). Triple negative breast cancer (TNBC) is an aggressive malignancy with high rates of p53 inactivation (~65–88%, (3–5)). TP53 mutations commonly arise in TNBC with missense mutations crippling its transcription factor function and nonsense mutations causing loss of p53 protein (6). Compared to other breast cancer types, TNBC patients have lower survival rates due to increased metastasis and relapse (7), and occur more frequently in African Americans (8). Because of their high p53 inactivation rates, compounds that inhibit p53 from binding to its negative regulator, MDM2, are ineffective in TNBC (9). Current standard-of-care treatment for TNBC is complex and dependent on several factors, but all approaches involve DNA-damaging chemotherapy that can result in cardiac and other toxicities (10). Unfortunately, efforts to treat this aggressive malignancy have made minimal progress in recent years, indicating innovative therapeutic strategies are needed.

Mouse developmental genetic studies showed deletion of Mdm2, a negative regulator of p53, was embryonic lethal unless p53 was concomitantly deleted (11,12). This established the view that if p53 was absent, cells were not impacted by Mdm2 loss. Mdm2/MDM2, an oncogene, is frequently overexpressed in cancer, including those with inactivated p53, as compared to normal cells, which have low levels of Mdm2/MDM2 (13,14). This suggested Mdm2/MDM2 may be important in cancer cells lacking functional p53. Recently, we reported that Mdm2 deletion induced apoptosis of p53-null murine sarcoma and T-cell lymphoma cells (15). However, whether a dependency on Mdm2 for cancer cell survival in mice translates to p53-inactivated human cancers, and particularly those with mutant p53, is unknown.

PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules composed of a targeting ligand and an E3 ubiquitin ligase recruiting ligand to induce selective degradation of specific cellular proteins (16). PROTACs provide pharmacological approaches for protein silencing, potentially mimicking genetic knockdown (17). Moreover, with their catalytic activity, PROTACs, should be able to be used at lower concentrations than small molecule inhibitors, making them less toxic. We sought to pharmacologically target MDM2 through the design and synthesis of a new MDM2-targeted PROTAC, YX-02–030, to evaluate the effects of MDM2 degradation on p53-inactivated TNBC. Our PROTAC potently binds MDM2 and recruits the von Hippel−Lindau (VHL) E3 ligase to initiate MDM2 degradation, and effectively killed p53 mutant or deleted TNBC cells in 2D and 3D culture models, patient explants, and in tumor xenografts through activation of the p53 family member TAp73. We show MDM2 is required for TNBC cell survival when p53 is inactivated, and PROTAC-targeted MDM2 degradation is a new potential therapeutic approach for this deadly cancer, demonstrating superiority to existing MDM2 inhibitors, which have been ineffective.

RESULTS

Design, synthesis, and protein binding capabilities of a new MDM2-targeted PROTAC

Although MDM2 levels are reported elevated in multiple human cancers that have inactivated p53 (14), it was unclear whether MDM2 was an essential, required protein for any human malignancy that had inactivated p53. Therefore, we designed and synthesized a new MDM2-targeting PROTAC, YX-02–030, to explore the requirements of MDM2 in human cancers, particularly those with high rates of TP53 mutation, such as TNBC (3–5). The MDM2-PROTAC, YX-02–030, replaces the 3-(methylsulfonyl)-propyl tail on the piperazine motif of the clinically available MDM2 inhibitor, RG7112, (18) with an acetic acid to generate RG7112D (Fig. 1A; chemical synthesis Supplementary Fig. S1A). An amide bond couples RG7112D to VHL-Amine, which is the VHL E3 ligase recruiting ligand, VH032 (ligand-7, (19), that is coupled to a hydrophilic tri-polyethylene glycol linker ((20); Fig. 1A). The resulting bi-functional molecule, YX-02–030, potently inhibited MDM2-p53 binding (HTRF IC₅₀ = 63±3nM; Fig. 1B), bound with high affinity to MDM2 (SPR K_D = 35nM; Fig. 1C; Supplementary Fig. S1B), inhibited VHL-HIF1α binding (HTRF IC₅₀ = 1350±181nM; Fig. 1B), and efficiently formed a ternary complex between GST-MDM2 and His-VHL (proximity-based AlphaScreen (21); Fig. 1D). Thus, our MDM2-targeted PROTAC has the binding and ternary complex formation abilities necessary to be an effective degrader.

Figure 1. Our MDM2-PROTAC (YX-02–030) binds MDM2 with high affinity, recruits VHL, and targets MDM2 for proteosome degradation.

A, Chemical structure of MDM2-PROTAC (synthesis in Supplementary Fig. S1A). B and C, MDM2:p53 and MDM2:VHL-HIF1α peptide binding inhibition determined by homogeneous time-resolved fluorescence (HTRF, triplicate, mean ± SEM, B) and surface plasmon resonance (SPR, triplicate, mean ± SD, C; see also Supplementary Fig. 1B) binding assays by the indicated compounds. Graphical data is representative of 3 separate experiments. D, AlphaScreen of ternary complex using GST-MDM2 and HIS-VHL; mean ± SD. E and F, Western blot analysis for the proteins indicated following a dose-titration (E, 16hr) and time course (F, 6μM) with the PROTAC or vehicle control (DMSO, -) in MDA-MB-231 (p53-mutant) and MDA-MB-436 (p53-delete) cells. G-K, Western blot analysis for the proteins indicated following treatment with the MDM2-PROTAC and/or the neddylation activating enzyme inhibitor MLN4924 (G), the proteasome inhibitor MG132 (H), RG7112 (I), VHL-Amine (J), the VHL small molecule inhibitor VH298 (K), or DMSO vehicle control (–).

Targeting MDM2 for proteasome-mediated degradation requires ternary complex formation

PROTACs work by forming a ternary complex consisting of the target protein, the PROTAC, and the recruited E3 ubiquitin ligase-E2 complex, causing ubiquitination of the target protein, marking it for 26S proteasome-mediated degradation (16). Our MDM2-directed PROTAC (YX-02–030) showed a concentration (Fig. 1E) and time-dependent (Fig. 1F) loss of MDM2 protein in TNBC cells with either mutated p53 (MDA-MB-231) or deleted p53 (MDA-MB-436). VHL protein levels remained unchanged, indicating MDM2, also an E3 ubiquitin ligase, was not ubiquitinating VHL (Fig. 1E, 1F). Since MDM2 can be phosphorylated, which can block antibody binding sites (22), we tested multiple MDM2 antibodies, and all showed loss of MDM2 protein with PROTAC exposure (Supplementary Fig. S1C). Moreover, loss of MDM2 protein was not due to decreased MDM2 mRNA levels, as they remained unchanged with PROTAC treatment (Supplementary Fig. S1D). Testing the contribution of the ubiquitin cascade with MLN4924, a NEDD8-activating enzyme inhibitor critical for ubiquitin transfer (23), showed it antagonized PROTAC-mediated MDM2 protein degradation in TNBC cells (Fig. 1G). Similarly, when we inhibited the proteasome with MG132, MDM2 protein levels were maintained despite MDM2-PROTAC exposure (Fig. 1H). Therefore, our MDM2-directed PROTAC targets MDM2 protein for 26S proteasome-mediated degradation in TNBC cells and this requires the ubiquitin cascade.

To determine whether the ternary complex was required for MDM2 degradation, we competed for binding to MDM2 and VHL by adding excess RG7112 or VHL-Amine, respectively. PROTAC-induced MDM2 degradation was prevented with either RG7112 or VHL-Amine (Fig. 1I and 1J, respectively). Analogous results were obtained using VH298, a VHL-specific small molecule inhibitor ((24); Fig. 1K). Thus, ternary complex formation of MDM2, the PROTAC, and VHL is required for PROTAC-induced MDM2 degradation.

MDM2-PROTAC activates wild-type p53 and is more effective than MDM2 inhibitors at inducing apoptosis of wild-type p53-containing breast cancer cells

To characterize the biological effects of our MDM2-PROTAC, we first tested it head-to-head with MDM2 inhibitors that prevent MDM2-p53 binding in p53 wild-type breast cancer cells with reported sensitivity to the MDM2 inhibitor Nutlin (25–27). Treatment of p53 wild-type MCF7 and DU4475 cells with MDM2 inhibitors (Nutlin, RG7112, RG7112D) or our MDM2-PROTAC resulted in the expected stabilization of MDM2 protein with the inhibitors and loss of MDM2 with the PROTAC (Fig. 2A). All compounds increased p53 protein levels (Fig. 2A) and significantly reduced cell survival in a concentration-dependent manner (Fig. 2B; Supplementary Fig. S2A). Of note, the IC₅₀ of our MDM2-PROTAC in MCF7 and DU4475 cells was lower than Nutlin (2.8μM and 2.3μM versus 11μM and 4μM, respectively) and lower than or equal to RG7112D and RG7112. Similarly, increased Caspase-3 activity was detected in MDM2-PROTAC-treated cells compared to those that received the MDM2 inhibitors at the same concentrations (Fig. 2C; Supplementary Fig. S2B). Over time, there was more cleaved PARP (Fig. 2D) and subG1 apoptotic DNA (Fig. 2E) with MDM2-PROTAC treatment compared to the MDM2 inhibitors. Furthermore, similar to Nutlin and its derivatives, MDM2-PROTAC treatment of MCF7 cells resulted in increased mRNA and protein levels of pro-apoptotic p53 target genes BAX, NOXA, PUMA (Fig. 2F and 2G, respectively).

Figure 2. MDM2-PROTAC is more effective than MDM2 inhibitors at activating p53 and killing p53 wild-type breast cancer cells.

MCF7 breast cancer cells (wild-type p53) were treated with the compounds indicated at 3μM, unless otherwise stated, or DMSO vehicle control. A, Western blotting for the indicated proteins and B, dose-titration curves (quadruplicate, MTT survival assay, 48hr) were performed. C-E, Apoptosis analyses, included Caspase-3 activity assay (C, triplicate), Western blotting for cPARP (D), and apoptotic subG1 DNA analysis (E, triplicate). F and G, expression of p53 target genes were evaluated by qRT-PCR (F, triplicate) and Western blotting (G). Acetyl choline receptor (AchR), served as a negative control for F. Cells were treated for 24hr for A, D, F and G. H, Mammosphere formation of MCF7 cells with the compounds indicated or DMSO control (-, triplicate, 72hr). I and J, MCF7 mammospheres were established and following treatment with the compounds indicated or DMSO control (–) survival/ATP production (I, quadruplicate, 72hr) and Caspase-3/7 activity (J, quadruplicate, 72hr) was determined. Representative images are shown (scale bar 300μm). Mean ± SD (B, C, E, H-J); mean ± SEM (F). For C, *P<0.0015, comparing each compound to DMSO vehicle control and **P<0.00021, comparing PROTAC to control compounds. For E, *P<0.0001, comparing each compound to DMSO vehicle control and **P<0.0001, comparing PROTAC to control compounds. For F, *P<0.0001, comparing each compound to DMSO vehicle control and **P<0.0001, comparing PROTAC to control compounds, except for PUMA (PROTAC vs RG7112, P=0.3714). For H, *P<0.00021, comparing each compound to DMSO vehicle control and **P<0.0016 and ***P<0.0001, comparing 3μM and 4μM PROTAC, respectively, to control compounds. For I, *P<0.0001, comparing each compound to DMSO vehicle control and **P<0.0001 and ***P<0.0001, comparing 3μM and 4μM PROTAC, respectively, to control compounds. For J, *P<0.0001, comparing each compound to DMSO vehicle control and **P<0.0001 and ***P<0.0001, comparing 3μM and 4μM PROTAC, respectively, to control compounds. 2-way ANOVA (C, E, F) and 1-way ANOVA (H-J).

We then evaluated whether the MDM2-PROTAC also impacts cell survival in 3D culture. MCF7 cells were treated with the MDM2-PROTAC or MDM2 inhibitors the same day they were placed in 3D culture. A significant decrease in the formation of mammospheres was observed in the PROTAC-treated cells compared to those exposed to MDM2 inhibitors or vehicle control (Fig. 2H; Supplementary Fig. S2C). For already established MCF7 mammospheres, our MDM2-PROTAC significantly decreased their survival (Fig. 2I) due to increased apoptosis (Caspase-3/7 activity) at half the concentration as the MDM2 inhibitors (Fig. 2J). Moreover, there was disaggregation of the mammospheres with MDM2-PROTAC treatment, but not with MDM2 inhibitor treatment (Fig. 2 images). Therefore, our MDM2-PROTAC activates p53 in p53 wild-type breast cancer cells, causing apoptosis in both 2D and 3D cultures and was equally or more potent than Nutlin and its derivatives.

p53-inactivated TNBC cells are sensitive to MDM2-PROTAC treatment

We next evaluated whether our MDM2-PROTAC could kill TNBC cells that had inactivated p53 by mutation or deletion. We tested the effects of our MDM2-PROTAC on TNBC lines (MDA-MB-231, HCC-1143, HCC-1395) with three different gain-of-function p53 point mutations (R280K, R248Q, R175H, respectively, (28–30)). All three mutant p53 lines showed sensitivity to the MDM2-PROTAC with a narrow IC₅₀ range (4.0–5.3μM), and no sensitivity to MDM2 inhibitors (Fig. 3A). Two p53-deleted TNBC cell lines (MDA-MB-436, MDA-MB-453) showed a dose-dependent decrease in survival with our MDM2-PROTAC with an IC₅₀ (4.5–5.5μM) similar to the p53-mutant lines, whereas RG7112 and RG7112D had no effect (Fig. 3A). These data reveal that in contrast to MDM2 inhibitors, p53 deletion or mutation does not impact sensitivity to our MDM2-PROTAC.

Figure 3. MDM2-PROTAC induces apoptosis of p53 mutant and deleted TNBC cells.

A, Dose-titration curves (quadruplicate, MTT or MTS survival assay, 48hr) of TNBC cell lines with p53 mutation (MDA-MB-231, HCC-1143, HCC-1395) or p53 deletion (MDA-MB-436, MDA-MB-453) treated with the compounds indicated or DMSO vehicle control. B, Dose-titration curves (quadruplicate, MTT survival assay, 48hr) of Mdm2^+/+p53^−/− and Mdm2^−/−p53^−/− mouse sarcoma cells treated with the indicated compounds; ABT-263 is a positive control. C, Inhibition of MDM2-PROTAC-induced death measured by competition assays with the indicated compounds in MDA-MB-231 cells (quadruplicate, MTT assay, 48hr). D, TNBC cells treated with their IC₅₀ (5μM for both) of MDM2-PROTAC, 5μM of control compounds, or DMSO vehicle control and MTT assays (quadruplicate) at 24hr intervals. E-G, MDA-MB-231 and MDA-MB-436 cells treated with MDM2-PROTAC (4μM) or DMSO vehicle control and apoptosis measured by Annexin-V positivity (E, triplicate), Caspase-3 activity (F, triplicate), and apoptotic subG1 DNA content (G, triplicate). H, Western blot analysis for the proteins indicated following treatment with MDM2-PROTAC and/or the compounds indicated, or DMSO vehicle control (–). β-ACTIN blots are the same as in Fig. 1H, 1J, and 1K. I-K, TNBC cells expressing three independent MDM2 shRNA or non-targeting control shRNA (shNT) and Western blots performed for the proteins indicated (I), MTT growth assays (quadruplicate, 24hr intervals, J), and apoptosis (K) measured by Annexin-V positivity (triplicate, left) and apoptotic subG1 DNA content (triplicate, right). Mean ± SD (A-G, J, K). For D-G, *P<0.0001, comparing PROTAC to control compounds and DMSO vehicle control. For J and K, *P<0.00051, comparing MDM2 shRNA to non-targeting control shRNA. 2-way ANOVA (D, J, K (left)), 1-way ANOVA (K (right)), and unpaired 2-tailed t-test (E-G). L, Spearman’s correlation of differential gene expression of RNA-seq data (triplicates) from MDA-MB-231 and MDA-MB-436 cells treated with MDM2-PROTAC versus expressing MDM2 shRNA, each first compared to their respective DMSO vehicle control or non-targeting shRNA control. Correlation coefficients (ρ) and P-values indicated.

Although there are no reports that the VHL recruiting ligand alone or as part of a PROTAC can function as a molecular glue, we tested the requirements of MDM2 and non-MDM2 lethal effects of our PROTAC. Because MDM2 is a highly conserved protein, we took advantage of p53-null mouse sarcoma cells from mice born with or without Mdm2 deletion. Mdm2^+/+p53^−/− sarcoma cells were as sensitive to our MDM2-PROTAC as the human TNBC cells (5.8μM IC₅₀; Fig. 3B), whereas Mdm2^−/−p53^−/− sarcoma cells were completely resistant to MDM2-PROTAC treatment, yet still sensitive to other compounds (e.g., ABT-263; Fig. 3B). Therefore, MDM2 must be present for our MDM2-PROTAC to be cytotoxic, indicating its lethal effect is not due to molecular glue.

To further test MDM2-targeted degradation as the cause for p53-inactivated TNBC cell death, we performed compound competition experiments as in Fig. 1I–K. Increasing concentrations of RG7112 or VHL-Amine to compete with the MDM2-PROTAC for binding MDM2 or VHL, respectively, blocked MDM2-PROTAC-induced TNBC cell death (Fig. 3C; Supplementary Fig. S3A). Analogous results were obtained with the VHL inhibitor VH298 (Fig. 3C; Supplementary Fig. S3A). Therefore, our MDM2-PROTAC kills p53-inactivated TNBC cells by targeting MDM2 and requires ternary complex formation.

Apoptosis induction and specificity of our MDM2-PROTAC verified with MDM2 knockdown in p53-inactivated TNBC cells

To characterize the type of cell death caused by our MDM2-PROTAC, we performed kinetic experiments using the IC₅₀ concentrations determined in Fig. 3A. The MDM2-PROTAC inhibited p53-mutant or deleted TNBC cell expansion over time (Fig. 3D; Supplementary Fig. S3B). Within 24 hours of MDM2-PROTAC treatment, there was significantly increased Annexin-V positivity (Fig. 3E; Supplementary Fig. S3C) and Caspase-3 activity (Fig. 3F; Supplementary Fig. S3D), both markers of apoptosis, and increased subG1 apoptotic DNA (Fig. 3G). PARP cleavage was also evident but was prevented when MDM2 degradation was inhibited with MG132, VHL binding to the PROTAC was blocked, or when VHL activity was inhibited (Fig. 3H; Supplementary Fig. S3E). These data show our MDM2-PROTAC induces p53-mutant or deleted TNBC cell apoptosis, and this requires ternary complex formation and MDM2 degradation.

To verify the apoptotic effects of our MDM2-PROTAC and its specificity for MDM2, we knocked down MDM2 with shRNA in both p53-mutant and deleted TNBC cell lines. Effective MDM2 knockdown (Fig. 3I) significantly reduced TNBC cell expansion (Fig. 3J) due to apoptosis; there was increased Annexin-V positivity (Fig. 3K), cleaved PARP (Fig. 3I), and subG1 apoptotic DNA (Fig. 3K). Because MDM2 knockdown mirrors the cytotoxic effects of our MDM2-PROTAC on p53-inactivated TNBC cells, this provides evidence supporting the MDM2 specificity of our PROTAC. Moreover, we conducted whole transcriptome analysis, and it showed there was a significant positive correlation between the genes with differential expression in MDA-MB-231 and MDA-MB-436 cells treated with the MDM2-PROTAC versus MDM2 knockdown (Spearman’s ρ=0.756, P=0 for MDA-MB-231 and ρ=0.836, P=0 for MDA-MB-436; Fig. 3L). These data provide independent evaluations of the specificity of our PROTAC for MDM2 and indicate it is targeting MDM2.

Reduced p53-inactivated TNBC clonogenic potential and increased mammosphere apoptosis with MDM2-PROTAC treatment

We next determined whether PROTAC-mediated degradation of MDM2 would alter the clonogenic potential of p53-inactivated TNBC cells by performing colony formation assays with MDA-MB-231 and MDA-MB-436 cells. Following MDM2-PROTAC treatment, there was a significant reduction in colony numbers with 1μM of MDM2-PROTAC (Fig. 4A). There was little/no effect with 2μM of MDM2 inhibitors, but ≤2 colonies formed with 2μM of MDM2-PROTAC (Fig. 4A).

Figure 4. Apoptosis induction of p53-inactivated TNBC cells in 2D and 3D cultures by the MDM2-PROTAC.

A and B, Colony formation assays (A, triplicate, 12 days) and mammosphere formation (B, triplicate, 72hr) of MDA-MB-231 and MDA-MB-436 cells in the presence of the indicated compounds or DMSO control (–); representative pictures shown (A). C-E, Formed mammospheres of MDA-MB-231 and MDA-MB-436 cells were treated with the compounds indicated or DMSO control (–), and area (C, n=10 mammospheres, relative to pre-treatment area, 72hr), real-time imaging (IncuCyte) and quantification of Caspase-3/7 activity (D, n=4–5 wells/compound), and survival/ATP assays (E, quadruplicate, 72hr) were performed; representative images shown; scale bar 300μm. Mean ± SEM (A-C and E). For A, *P<0.0001 and **P<0.0001, comparing 1μM and 2μM PROTAC, respectively, to control compounds and DMSO vehicle control. For B, *P<0.0046, **P<0.0001, and ***P<0.0001, comparing 0.5μM, 1μM, and 2μM PROTAC, respectively, to control compounds and DMSO vehicle control. For C, *P<0.0001 and **P<0.0001, comparing 2μM and 4μM/6μM PROTAC, respectively, to control compounds and DMSO vehicle control. For E, *P<0.0001 and **P<0.0001, comparing 3μM and 4μM PROTAC, respectively, to control compounds and DMSO vehicle control. 1-way ANOVA (A-C and E).

We then evaluated in 3D culture whether our MDM2-PROTAC was effective at preventing TNBC cell mammosphere formation and eliminating already formed mammospheres. For both MDA-MB-231 and MDA-MB-436 cells, significantly fewer mammospheres formed at each MDM2-PROTAC concentration tested, compared to the MDM2 inhibitors and vehicle control (Fig. 4B). In already-established mammospheres, there was a significant decrease in mammosphere area following MDM2-PROTAC treatment (Fig. 4C). Notably, a 3-fold lower concentration of MDM2-PROTAC than the MDM2 inhibitors reduced mammosphere area by 49.5% and 43.8% in MDA-MB-231 and MDA-MB-436 mammospheres, respectively, and equal concentrations reduced mammosphere area even more (Fig. 4C). This reduction was due to apoptotic cell death, as we detected significantly increasing Caspase-3/7 activity over time (Fig. 4D) and decreased survival (Fig. 4E) in the mammospheres following MDM2-PROTAC treatment compared to the MDM2 inhibitors. Together, our data provide strong evidence that our MDM2-PROTAC induces apoptosis in p53-inactivated TNBC cells in both 2D and 3D culture models.

MDM2-PROTAC is stable and effectively kills TNBC cells in vivo

Prior to testing our MDM2-PROTAC on tumors in vivo, we performed mouse liver microsome and pharmacokinetic (PK) studies to determine its stability in vivo. Our MDM2-PROTAC was moderately metabolically stable with a 25.9-minute half-life (Fig. 5A) and had excellent in vivo stability with plasma levels stable over 6 hours in mice after a single 10mg/kg intraperitoneal dose (Fig. 5B). Due to its stability in vivo, we then evaluated the effectiveness of our MDM2-PROTAC at killing TNBC tumors in mice. Xenografts of MDA-MB-231 and MDA-MB-436 TNBC cells grew to ~80mm³, and then tumor size-matched mice were treated with our MDM2-PROTAC, RG7112D control compound, or vehicle control. Fourteen days of MDM2-PROTAC treatment significantly extended mouse survival (Fig. 5C) and decreased tumor volume (Fig. 5D; Supplementary Fig. S4A), compared to control mice for both xenograft models. To confirm our MDM2-PROTAC was hitting its target (MDM2) in the TNBC tumors, we harvested tumors from a cohort of mice after 72 hours of MDM2-PROTAC treatment. Mice that received MDM2-PROTAC showed loss of MDM2 protein (Fig. 5E) and increased cleaved PARP (Fig. 5E), Annexin-V positivity (Fig. 5F), Caspase-3 activity (Fig. 5G), subG1 apoptotic DNA (Fig. 5H), and non-viable cells (Fig. 5I). Notably, no signs of overt toxicity in immune-competent C57Bl/6 mice (Supplementary Fig. S4B, S4C) or immune-deficient mice from the xenograft experiments (Supplementary Fig. S4D–F) were observed following MDM2-PROTAC treatment. Specifically, mouse weight was maintained and complete blood counts and the histology and cellular content of the spleen, bone marrow, and intestine were normal (Supplementary Fig. S4). Therefore, our MDM2-PROTAC showed clear in vivo efficacy against p53-inactivated TNBC tumors and no obvious toxicity to normal tissues.

Figure 5. MDM2-PROTAC kills p53-inactivated TNBC cells in vivo.

A, Liver microsomal stability evaluated for the MDM2-PROTAC and control compounds. Midazolam served as an unstable control. B, Pharmacokinetic analysis of MDM2-PROTAC in plasma in mice (n=3/timepoint, single 10mg/kg intraperitoneal injection of MDM2-PROTAC). C-I, MDA-MB-231 and MDA-MB-436 cells were injected (subcutaneous) into one flank of nude mice and allowed to form palpable tumors (approximately 80mm³). Mice (randomized into tumor-size matched groups) were treated (intraperitoneal injection) with MDM2-PROTAC, RG7112D, or vehicle control at 50mg/kg once daily for 14 days (treatment start, green arrow and stop, red arrow). Kaplan-Meier survival analysis (C) and tumor volume (D; individual, left and averaged, right) were evaluated overtime. E-I, After 72hr of treatment, tumors (n=3/group) were harvested and protein expression evaluated by Western blotting (E; -, vehicle control), Annexin-V positivity (F), Caspase-3 activity (G), apoptotic subG1 DNA content (H), and viability by Trypan Blue dye exclusion (I). Mean ± SD (B, D (right), F-I). Log-rank tests for C, and longitudinal tumor growth analysis using 2-way ANOVA with Bonferroni correction for D; P-values indicated. *P<0.0021 for F, *P<0.0001 for G, *P<0.0147 for H, and *P<0.0014 for I were determined using unpaired two-tailed t-tests, comparing PROTAC to vehicle control.

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Because clinical trials of RG7112 showed hematopoietic cell toxicities (31,32), to further investigate the lack of MDM2-PROTAC-induced cytotoxicity of normal cells we tested whether p53 was stabilized and its target genes induced in normal hematopoietic cells upon MDM2-PROTAC treatment. First, following three days of treatment (same regimen as Fig. 5E–I), splenocytes and bone marrow cells from C57Bl/6 mice were evaluated. In the mice that were irradiated (positive control), p53 was stabilized and there were increased protein and mRNA levels of p53 targets (BAX, NOXA, and PUMA), but not in the MDM2-PROTAC treated mice (Supplementary Fig. S4G, S4H). Secondly, we tested normal human CD34+ hematopoietic cells. CD34+ cell viability remained largely unaffected by MDM2-PROTAC treatment, but viability significantly decreased after RG7112 treatment and to a lesser extent with RG7112D (Supplementary Fig. S4I; MDM2-PROTAC treated MDA-MB-231 TNBC cells were a positive control). Also, in CD34+ cells, mRNA levels of the p53 target genes, BAX, NOXA, and PUMA were unchanged following MDM2-PROTAC treatment, but were significantly increased with RG7112 and less strongly with RG7112D (Supplementary Fig. S4J). Thus, our MDM2-PROTAC shows specificity for cancer cells, as it did not activate p53-mediated apoptotic gene upregulation in normal mouse or human hematopoietic cells. However, RG7112, which is known to cause bone marrow cell toxicity in humans (31,32), did negatively affect human CD34+ cells by activating p53.

Targeted MDM2 degradation induces apoptosis in patient-derived TNBC explants

To assess the effects of our MDM2-PROTAC on TNBC patient samples, fresh, surgically-resected tumor from five TNBC patients was obtained, and 80% were from Black/African American patients (Supplementary Table S1). p53 is mutated in the vast majority of TNBC (3–5). After sequencing TP53 in the five TNBC samples we were provided, all had TP53 missense mutations with two having the same hot-spot (R248Q) p53 mutation and one that had 3 TP53 mutations (Fig. 6A). While preserving tumor architecture, pieces of each patients’ tumor were placed into explant cultures (33,34) and subjected to treatment with our MDM2-PROTAC and vehicle control, and when enough tissue was provided, also RG7112D. Within 4 days of treatment, MDM2 protein was lost in the patient tumors that received the MDM2-PROTAC compared to vehicle and RG7112D controls, and as expected, no change in VHL protein (Fig. 6B). MDM2-PROTAC-treated patient tumors were undergoing apoptosis, as those explants showed increased levels of cleaved Caspase-3 and cleaved PARP by Western blot (Fig. 6B). Immunohistochemistry also showed a significantly increased number of cleaved Caspase-3 positive tumor cells in MDM2-PROTAC-treated explants, but normal breast epithelial and stromal cells were unaffected (Fig. 6C). Thus, the data from the patient-derived TNBC explants show that our MDM2-PROTAC effectively and specifically kills TNBC patient tumor cells.

Figure 6. TP53 mutant TNBC patient-derived explants undergo apoptosis following treatment with the MDM2-PROTAC.

A, TP53 sequenced in five fresh TNBC patient samples and used in explant and 3D mammosphere cultures. B and C, TNBC patient-derived explants were treated with the compounds indicated and Western blotting (B) and immunohistochemistry (IHC) of FFPE sections for cleaved Caspase-3 (C, n=3 paired patient samples) were performed. Representative images of H&E and cleaved Caspase-3 (CC3) IHC shown, including normal breast epithelium treated with MDM2-PROTAC; scale bar 300μm. D and E, Mammosphere cultures were established from TNBC patient samples, treated with the indicated compounds, and live-cell detection of Caspase-3/7 activity (D, quadruplicate) and mammosphere survival/ATP production (E, quadruplicate, 96hr) were measured. Normal breast epithelium from a patient that formed loose aggregates was also treated with MDM2-PROTAC. For comparison, values for TNBC-4 and the normal breast mammospheres are relative to DMSO vehicle control (E, right graph). Representative images shown (RG, RG7112 and RG-D, RG7112D); scale bar 300μm. Mean ± SD (C-E). For C, *P=0.0477 (unpaired two-tailed t-test), comparing PROTAC to DMSO. For D and E, *P<0.0001 (2-way and 1-way ANOVA, respectively), comparing PROTAC to control compounds and DMSO vehicle control.

For three of the patient TNBC tumor samples, we had enough tissue to also establish 3D mammosphere cultures, and for one of these patients, we also received normal breast epithelial tissue that generated loose cell clusters. Our MDM2-PROTAC significantly increased Caspase-3/7 activity (Fig. 6D) and reduced survival (Fig. 6E) of the mammospheres from all three patient tumors, whereas MDM2 inhibitors had no effect. The normal breast epithelial clusters remained largely unaffected by MDM2-PROTAC treatment (Fig. 6D, 6E). These data provide significant evidence that targeting MDM2 for degradation may be a viable, non-toxic therapeutic strategy for TNBC.

TAp73 is induced and required for apoptosis upon MDM2-targeted loss in p53-inactivated TNBC cells

To gain insight into the mechanism by which our MDM2-PROTAC was killing p53-mutant and deleted TNBC cells, we evaluated RNA-sequencing data on cells treated with MDM2-PROTAC and cells expressing MDM2 shRNA and each of their controls. As expected, evaluation of gene signatures from the Hallmark database (35) that were significantly enriched showed genes linked to apoptosis in both cell lines whether they were treated with the MDM2-PROTAC or MDM2 was knocked down (>2 fold-change, FDR<0.05; Fig. 7A; Supplementary Fig. S5A). Additionally, our analysis also revealed p53 pathway gene signatures were also significantly enriched (>2 fold-change, FDR<0.05; Fig. 7A; Supplementary Fig. S5A). More than 25 genes targeted by the p53 family, as reported in the IARC TP53 Database (36), showed significantly elevated expression in both p53-mutant and deleted TNBC cells with MDM2 degradation or knockdown (FDR<0.05; Fig. 7B; Supplementary Fig. S5B). Notably, there was a highly significant correlation between p53 family-targeted genes with differential expression in MDA-MB-231 and MDA-MB-436 cells treated with the MDM2-PROTAC versus MDM2 knockdown (Spearman’s ρ=0.873, P=0 for MDA-MB-231 and ρ=0.931, P=0 for MDA-MB-436; Fig. 7C; Supplementary Fig. S5C), further illustrating the specificity of our PROTAC for MDM2.

Figure 7. TAp73 is activated by the MDM2-PROTAC and mediates apoptosis of p53-inactivated TNBC cells.

A-C, RNA-seq (triplicates) was performed on MDA-MB-231 cells treated with the MDM2-PROTAC (6μM, 16hr) or DMSO vehicle control or expressing MDM2 shRNA or non-targeting control shRNA (48hr). A, Pathway enrichment analysis using Hallmark gene signatures. B, Heatmap of p53 family target genes of individual samples (3 each). C, Spearman’s correlation of differentially expressed p53 family target genes between PROTAC-treated relative to DMSO vehicle control and MDM2 shRNA relative to non-targeting shRNA control; Spearman’s correlation coefficient (ρ) and P-value indicated. D, qRT-PCR analysis (triplicate, 6μM, 16hr) to validate RNA-seq results. E-H, Western blotting performed for the proteins indicated following treatment with MDM2-PROTAC, control compounds, or DMSO vehicle control (–) of MDA-MB-231 cells growing in culture (E, 6μM, 16hr), MDA-MB-231 subcutaneous tumors harvested 72hr after treatment began (G, top, same β-ACTIN blot as Fig. 5E), TNBC patient-derived explants (G, bottom, same β-ACTIN blots as Fig. 6B), following knockdown of MDM2 with two shRNA or non-targeting shRNA (shNT) control in MDA-MB-231 cells (F), or following treatment with the PROTAC (6μM, 16hr) in MDA-MB-231 cells pre-treated (1hr, 10μM) with RG7112, VHL-Amine, or VH298 (H). I, TAp73 ChIP was performed with MDA-MB-231 cells following treatment with the MDM2-PROTAC (6μM, 16hr) or DMSO vehicle control, and enrichment of TAp73 (triplicate; first normalized to input DNA then IgG control) was determined at the loci indicated. J, MDA-MB-231 cells pre-treated (1hr, 10μM) with MG132 were treated with MDM2-PROTAC (6μM, 16hr) or DMSO vehicle control and MDM2 (top) and TAp73 (bottom) were immunoprecipitated and proteins Western blotted. K, Whole cell lysates (WCL) from MDA-MB-231 cells treated with MDM2-PROTAC, RG7112D, RG7112 (all at 6μM, 16hr) or DMSO vehicle control were Western blotted (left), or TAp73 was immunoprecipitated and then proteins Western blotted (right). L and M, After 48hr, MDA-MB-231 cells expressing two independent TAp73 shRNA or non-targeting shRNA (shNT) control were treated with MDM2-PROTAC or DMSO vehicle control (6μM, 16hr). p53/TAp73 target genes were evaluated by Western blotting (L) and qRT-PCR (M, triplicate). N, MTT assay (quadruplicate, 24hr intervals) of MDA-MB-231 cells expressing two independent TAp73 shRNA or non-targeting shRNA (shNT) control and treated with MDM2-PROTAC (4μM) or DMSO vehicle control. Mean ± SEM (D, I, M) and mean ± SD (N). For D and I, *P<0.0001 (unpaired two-tailed t-tests), comparing PROTAC to DMSO vehicle control or MDM2 shRNA to non-targeting control shRNA. For M, *P<0.0015, comparing non-targeting shRNA control with PROTAC to DMSO vehicle control and **P<0.0012, comparing TAp73 shRNA with PROTAC to non-targeting shRNA control with PROTAC; 2-way ANOVA. For N, *P<0.00081, comparing non-targeting control shRNA with PROTAC to DMSO vehicle control and **P<0.0031, comparing TAp73 shRNA with PROTAC to non-targeting control shRNA with PROTAC; 2-way ANOVA. For E-G and L, long exp is long exposure.

To independently validate our RNA-seq results, we performed qRT-PCR on a subset of the genes regulated by the p53 family that mediate apoptosis. Following MDM2 degradation or MDM2 knockdown in MDA-MB-231 and MDA-MB-436 cells, levels of apoptotic genes BAX, NOXA, PUMA, AEN, APAF1, TP53I3, and PIDD1 were significantly increased in both TNBC cell lines compared to controls (Fig. 7D; Supplementary Fig. S5D).

MDM2 can bind and regulate p53 family members p73 (37–40) and p63 (41,42), but the conditions in which this occurs, particularly in p53-inactivated cells, remain unresolved. Because the transcriptionally active forms of p73 (TAp73) and p63 (TAp63) are capable of transactivating most of the same genes as p53 (6), we evaluated their expression. Upon MDM2-PROTAC treatment or MDM2 knockdown, levels of TAp73 protein increased in all five TNBC cell lines assessed (Fig. 7E, 7F; Supplementary Fig. S6A–C); however, TAp63 was lowly expressed and its levels remained unchanged (Fig. 7E, 7F; Supplementary Fig. S6A, S6B). Additionally, levels of TAp73 protein, but not TAp63, were increased in the xenograft tumors harvested 72 hours after PROTAC treatment and in the TNBC patient-derived explants following MDM2-PROTAC treatment (Fig. 7G; Supplementary Fig. S6C). In contrast, MDM2 inhibitor treatment did not result in increased TAp73 levels. Note that to effectively detect TAp73 and TAp63, proteins were isolated under conditions designed to extract transcription factors, which tend to be positively charged and/or chromatin-bound (Supplementary Fig. S6D). Levels of ΔNp73, which lacks the N-terminal transactivation domain and can inhibit TAp73 (43), in MDA-MB-231 and MDA-MB-436 cells were low and unaffected by MDM2 degradation or knockdown (Fig. 7E, 7F; Supplementary Fig. S6A, S6B). Increased TAp73 protein was not due to increased transcription, as TAp73 mRNA levels were unaltered upon MDM2 loss (Supplementary Fig. S6E). Both mRNA (Fig. 7D; Supplementary Fig. S5D) and protein (Fig. 7E, 7F; Supplementary Fig. S6A, S6B) levels of TAp73 apoptotic targets were upregulated with MDM2 loss in the TNBC cell lines, suggesting TAp73 was activated. Additionally, we evaluated TAp73 after PROTAC binding to MDM2 or VHL was blocked with excess RG7112 or VHL-Amine, respectively, or when VHL was inhibited with VH298. TAp73 protein was only stabilized, its apoptotic target genes upregulated, and apoptosis induced (cPARP) when the PROTAC was able to successfully target MDM2 for degradation, but not when the ternary complex was disrupted (Fig. 7H; Supplementary Fig. S6F). Moreover, chromatin immunoprecipitation of TAp73 after MDM2-PROTAC treatment, showed significant enrichment of TAp73 at the promoters of its apoptotic target genes (BAX, NOXA, PUMA, AEN, APAF1, TP53I3, PIDD1), but not at the promoter of AchR, which is not targeted by p53/TAp73 ((44), Fig. 7I; Supplementary Fig. S6G). These data indicate that MDM2-PROTAC treatment causes TAp73 to be stabilized and transcriptionally activated.

To better understand how MDM2 loss results in TAp73 stabilization and transcriptional activation, we began by assessing whether our MDM2-PROTAC binding in the p53/p73 binding pocket of MDM2 disrupts interactions with TAp73 in p53-inactivated TNBC cells. We performed immunoprecipitations of both MDM2 and TAp73 in the presence of the MDM2-PROTAC or vehicle control in p53-mutant and deleted TNBC cells. The proteasome inhibitor MG132 was included to prevent proteasomal degradation of MDM2. In the MDM2-PROTAC-treated samples, there was markedly less TAp73 associated with MDM2, in both MDM2 and TAp73 immunoprecipitations (Fig. 7J; Supplementary Fig. S6H), indicating the PROTAC was interfering with MDM2:TAp73 binding. Because TAp73 was binding to MDM2 in the TNBC cells, disruption of this interaction would be expected since a derivative of RG7112 is the MDM2-targeting molecule in the MDM2-PROTAC and binds tightly to the p53/p73 binding pocket of MDM2 (Fig. 1B). However, for TAp73 to be an active transcription factor, our data indicate that it needs more than losing its association with MDM2. Since TAp73 requires specific post-translational modifications to be transcriptionally active, we evaluated phosphorylation of tyrosine-99 in TAp73, the site phosphorylated by c-Abl and a requisite to be activated, leading to TAp73 stabilization (45). Following treatment of MDA-MB-231 and MDA-MB-436 cells with our MDM2-PROTAC, TAp73 Tyr-99 was phosphorylated, but not after treatment with RG7112D or RG7112 (Fig. 7K; Supplementary Fig. S6I). In addition, c-Abl was phosphorylated at Tyr-412, a mark of its activation, in the cells exposed to MDM2-PROTAC, but not with RG7112D or RG7112 (Fig. 7K; Supplementary Fig. S6I). TAp73 Tyr-99 phosphorylation is a signal that should call in the CBP/p300 lysine acetyltransferase complex, which is necessary for TAp73 to become transcriptionally activated (45). Immunoprecipitation of TAp73 showed its association with CBP and p300 and acetylation of TAp73 after MDM2-PROTAC treatment in MDA-MB-231 and MDA-MB-436 cells, but not following treatment with the MDM2 inhibitors (Fig. 7K; Supplementary Fig. S6I). Together these data show that in p53-inactivated TNBC cells, MDM2 degradation results in the transcriptional activation of TAp73 and that simply releasing TAp73 from MDM2 is insufficient to induce TAp73-mediated transcription.

To assess the requirements of p73 in mediating the effects of the MDM2-PROTAC, we knocked down TAp73 using two independent shRNA in p53-mutant and deleted TNBC cells. These two shRNAs were TAp73-specific, as they did not impact levels of mutant p53, TAp63, or ΔNp73 (Fig. 7L; Supplementary Fig. S6J). Although MDM2 protein was lost following MDM2-PROTAC treatment, knockdown of TAp73 in both TNBC lines prevented upregulation of its apoptotic target genes (Fig. 7M; Supplementary Fig. S6K), which was also reflected at the protein level (Fig. 7L; Supplementary Fig. S6J). TAp73 knockdown also largely rescued the decrease in cell growth (Fig. 7N; Supplementary Fig. S6L) and the cleavage of PARP (Fig. 7L; Supplementary Fig. S6J), resulting from PROTAC-mediated MDM2 degradation. Collectively, our results indicate that our MDM2-PROTAC effectively targets MDM2 for degradation, activating TAp73-dependent apoptosis in p53-inactivated TNBC, and provides a future avenue for treatment of this deadly cancer.

DISCUSSION

Based largely on developmental data with p53 and Mdm2 knockout mice, it was believed that once p53 was inactivated, cells no longer needed Mdm2 (11,12). However, our data here challenge this dogma, revealing that MDM2 is required in p53-mutant and deleted human cancer cells, specifically TNBC cells, for their continued growth and survival. We have capitalized on this identification of a targetable vulnerability in p53-inactivated TNBC by designing and synthesizing a new MDM2-PROTAC. The MDM2-PROTAC, YX-02–030, specifically targets MDM2, is stable, and eliminates TNBC cells in 2D and 3D cultures, patient TNBC explants, and xenografts in mice, while sparing normal cells, by activating TAp73, which induces apoptosis (Supplementary Fig. S7). With Blacks/African Americans having an increased frequency of TNBC (8), 80% of our patient explants were from this demographic. Our results open an entirely new avenue for treating patients with aggressive TNBC that harbor inactivated p53 that has allowed them to evade many therapies. Moreover, because PROTACs have catalytic activity, this allows them to be able to be used at lower concentrations than conventional small molecule inhibitors, which should make our MDM2-PROTAC less toxic.

PROTACs require high affinity binding to the targeted protein and recruitment of an E3 ligase that ubiquitinates the target, resulting in proteosome-mediated degradation of the protein (16). Both HTRF and SPR analyses showed high affinity binding of our PROTAC to MDM2 and binding to VHL, and AlphaScreen analysis confirmed formation of the ternary complex. Using inhibitors and competition assays, we showed MDM2 underwent proteosome-mediated degradation that required binding to MDM2 and recruitment of VHL. We utilized the VHL recruiting ligand, which has not been reported to function as a molecular glue, unlike the Cereblon recruiting ligand (46–48). Our MDM2-PROTAC required MDM2 and did not function as a molecular glue, and formation of the ternary complex did not result in MDM2-mediated ubiquitination and degradation of VHL. There are multiple possible reasons why VHL is not affected by our MDM2-PROTAC; for example, the length and flexibility/rigidity of the linker that impacts the orientation of the ternary complex only allowing MDM2 to be ubiquitinated by VHL (49), and the inaccessibility of the three lysines of VHL when complexed with the proteins necessary to transfer ubiquitin to MDM2 (50). Importantly, comparisons between our MDM2-PROTAC and MDM2 knockdown in every assay consistently showed analogous results. Moreover, Spearman’s correlation coefficients were strongly positive in two p53-inactivated TNBC lines comparing globally differentially expressed genes after MDM2-PROTAC treatment to MDM2 knockdown. Taken together, the data indicate our MDM2-PROTAC specifically targets MDM2 for degradation.

Previous reports, largely limited to acute lymphoblastic leukemia, have employed compounds being asserted as MDM2-specific degraders, but oddly, they only kill wild-type p53 cancer cells and not p53-inactivated cancer cells (47,48,51–53). This is inconsistent with multiple lines of evidence that we and others have obtained. Our evaluation of the effects of Mdm2 deletion and MDM2 knockdown in two species (mouse and human) resulted in data mirroring the data we obtained with our MDM2-PROTAC, showing that loss of MDM2 kills p53-inactivated cancers. Moreover, inhibiting MDM2 E3 ubiquitin ligase activity with the compound MEL23 was recently shown to induce death in some p53-mutant and deleted cancer cells (54), suggesting MDM2 E3 ligase function is necessary for cancer cell survival. One compound designed as an MDM2-PROTAC used a Cereblon recruiting ligand and was shown to function as a molecular glue, independent of the MDM2 ligand, and therefore was not an MDM2-PROTAC (48). Cereblon has also been shown to bind and degrade neosubstrates (55), so PROTACs that use Cereblon may also lead to degradation of these neosubstrates that may contribute to the effects observed. PROTAC studies have used a phosphorylation sensitive MDM2 antibody that does not bind MDM2 when it is phosphorylated at that site (22,47,48,53), possibly masking its presence; we tested multiple MDM2 antibodies to rule this out. It is also possible that there are cancer cell-type or context-specific effects for MDM2-PROTACs. For example, the E3 ligase being recruited could have very different expression in different cell types and conditions. While further investigation will be needed to resolve the differences between compounds, our data consistently show that MDM2 is required for cancer cell survival and that targeted MDM2 degradation with our MDM2-PROTAC kills TNBC.

The multiple MDM2 inhibitors available were designed to bind MDM2 with high affinity, disrupt p53:MDM2 interactions, and were shown to stabilize MDM2 protein, leading to significantly increased MDM2 levels (18,56–61). Use of these inhibitors in patients have resulted in neutropenia and thrombocytopenia (32,62), raising concerns for their use. The high levels of MDM2 caused by these inhibitors, which engages both p53-dependent and p53-independent MDM2 functions (15,59,63–65), are thought to contribute to the toxic effects observed in patients. A distinct advantage of our MDM2-PROTAC is that it degrades MDM2, preventing the toxic buildup of MDM2 observed with current inhibitors (18,59,62,66). Moreover, since PROTACs function in a catalytic manner, it is expected that lower doses than those used for MDM2 inhibitors would reduce possible cytotoxicity. Concerns about globally or locally activating wild-type p53 in normal tissues with MDM2 degradation is significantly reduced with our data showing normal human breast epithelial or stromal cells and CD34+ hematopoietic cells were not affected by our MDM2-PROTAC and similarly in mice, there was no overt toxicity detected in hematopoietic or other tissues. Of note, we designed our PROTAC to recruit VHL, and VHL levels are especially low in platelets compared to cancer cells (67) and would therefore, lower the risk of patients developing thrombocytopenia, as is observed with MDM2 inhibitors (31,32). In contrast to RG7112, our MDM2-PROTAC caused no toxicity to human CD34+ bone marrow cells, providing further evidence that our PROTAC should be safer than the MDM2 inhibitors have shown to be. Moreover, recently, a compound (PC14586) that stabilizes the Y220C destabilizing mutant p53 was tested in Phase I trials in patients with solid tumors. Surprisingly, Li Fraumeni patients with this germline p53 mutation were also included in this testing cohort, and notably, did not show increased toxicity to this compound (68). These results indicate that the normally low levels of wild-type p53 in tissues are not activated with loss of MDM2 or stabilization of a destabilizing mutant p53, most likely due to another stress needed to activate p53.

Our MDM2-PROTAC effectively killed both p53-mutant and deleted TNBC cells in vitro and in vivo with a similar IC₅₀ for both. RNA-seq data in both p53 mutant and deleted TNBC revealed the effects of our MDM2-PROTAC was an apoptotic mechanism mediated by TAp73 transcriptional up-regulation of pro-apoptotic genes. MDM2 binding TAp73 inhibits its transcriptional function by preventing it from binding chromatin, and is not thought to ubiquitinate TAp73 as it does p53 (37,39,40,63), but this is controversial (69,70). Instead, MDM2 can regulate TAp73 by blocking it from binding to transcriptional cofactors (39). We determined that MDM2 degradation, but not MDM2 inhibition, resulted in the phosphorylation of TAp73, which stabilizes it, and the association of TAp73 with CBP/p300 and its acetylation. Therefore, our MDM2-PROTAC results in an increased amount of TAp73 present at the chromatin competent to transcribe genes, which our data showed. Specifically, data from multiple human TNBC cell lines in culture, xenograft tumors in vivo, and TNBC patient explants treated with our MDM2-PROTAC all showed TAp73 activation. Similarly, we also observed p73 activation in p53-null mouse cancers when Mdm2 was deleted (15). Although it has been reported that specific p53 gain-of-function mutants can inhibit TAp73 transcriptional activity (71–73), that did not appear to occur to an extent that would block the induction of TAp73-mediated transcription of apoptotic target genes and the resulting apoptosis we observed. In addition, reduced levels of TAp73 via knockdown largely blocked the effect of our MDM2-PROTAC, suggesting that loss of p73 could confer resistance to our PROTAC. Collectively, our data show that in both human and mouse cancer cells with p53-inactivation, TAp73 is activated with MDM2/Mdm2 loss by shRNA, our PROTAC, or deletion, and that TAp73 compensates for the lack of functional p53 under these conditions in TNBC cells.

The failure of current preclinical cancer models to predict patient responses continues to significantly impact the ability to leverage targeted therapies for precision medicine (74). By utilizing patient-derived explants and 3D culture models of cell lines and patient-derived mammospheres, we observed that our MDM2-PROTAC killed p53-inactivated TNBC cells, including those from patients that had relapsed, and not normal cells. We also determined that our MDM2-PROTAC induced apoptosis of p53 wild-type breast cancer cells, indicating applicability of our PROTAC beyond p53-inactivated cancers. Currently, two PROTACs, ARV-110 (NCT03888612) and ARV-471 (NCT04072952), that target the androgen receptor and the estrogen receptor, respectively, were determined safe and have advanced to phase II clinical trials (75,76). There are also multiple other PROTACs that target proteins, such as BTK (NX-2127), BCL-X_L (DT2216), IRAK4 (KT-474), BRD9 (CFT8634/FHD-609), and STAT3 (KT-333), that are in phase I trials for different cancer types (75) and more on the way to the clinic, highlighting that PROTACs are a rapidly growing treatment approach. Because TNBC typically grows and spreads quickly, has few treatment options, and patients tend to have a worse prognosis, our preclinical data have highlighted our MDM2-PROTAC as a new potential therapeutic approach for treating p53-inactivated TNBC.

MATERIALS AND METHODS

Synthesis of the MDM2 PROTAC YX-02–030

Detailed synthetic procedures, NMR-spectra, and characterization are in Supplementary Methods.

Compounds

Compounds were purchased from SelleckChem: RG7112 (#S7030), MG132 (#S2619), ABT-263/navitoclax (#S1001), MLN4924 (#S7109), VH298 (#S8449), and Doxorubicin (#S1208) or Sigma (Nutlin-3 #N6287 and Etoposide #E1383). YX-2–23 (RG7112 derivative) and VHL-Amine were synthesized (see Supplementary Methods for details).

HTRF binding assays

For MDM2:p53 HTRF binding assays, GST-MDM2 and HIS-p53 were expressed in E. coli and purified by affinity chromatography. pGEX-4T-MDM2-WT (RRID:Addgene_16237) and human p53-(1–393) (RRID:Addgene_24859) were from Addgene. HTRF assays contained GST-MDM2, HIS-p53, anti-GST-Tb HTRF donor (RRID:AB_2927626) and anti-HIS-d2 HTRF acceptor (RRID:AB_2884027) antibodies (PerkinElmer-CisBio). HTRF assays testing the inhibition of VHL peptide binding to VHL complex contained VHL complex (R&D Systems), biotinylated VHL peptide, anti-HIS-Tb-Gold HTRF donor antibody (RRID:AB_2716834), and anti-streptavidin-d2 HTRF acceptor (RRID:AB_2928111; PerkinElmer-CisBio). IC₅₀ values were determined using nonlinear regression with four-parameter dose-response curve equation. Details are in Supplementary Methods.

SPR binding kinetics and affinity analysis

Association and dissociation rate constants for MDM2-PROTAC and control compounds were determined using a Biacore-T200 SPR instrument using HIS-SUMO-MDM2 (expressed and purified) immobilized on a multidentate linear carboxylate high affinity nickel chip (Xantec-Bioanalytics). Kinetic data were globally fit to a single site binding model using Biacore evaluation software (RRID:SCR_008424). Details are in Supplementary Methods.

Alpha Screen for ternary complex formation

GST-MDM2 and HIS-VHL complex were pre-incubated with AlphaScreen donor or acceptor beads, respectively. Dose-response assays were initiated by combining the GST-MDM2/AlphaScreen donor bead mix with the HIS-VHL complex/AlphaScreen acceptor bead mix. AlphaScreen signals were measured using the ClarioStar plate reader (BMG-Labtech). Data were fit to a bell-shaped dose-response curve equation in GraphPad Prism (RRID:SCR_002798). Details are in Supplementary Methods.

Cell culture

DU4475 (HTB-123; RRID:CVCL_1183), MCF7 (HTB-22; RRID:CVCL_0031), MDA-MB-231 (HTB-26; RRID:CVCL_0062), MDA-MB-436 (HTB-130; RRID:CVCL_0623), MDA-MB-453 (HTB-131; RRID:CVCL_0418), HCC-1143 (CRL-2321; RRID:CVCL_1245), HCC-1395 (CRL-2324; RRID:CVCL_1249), and HCC-1937 (CRL-2336; RRID:CVCL_0290) cell lines were obtained from ATCC and cultured as they directed. Cell lines were authenticated using short tandem repeat profiling. Murine sarcoma primary cell cultures were derived from sarcoma spontaneously arising in Mdm2^−/−p53^−/− (from Dr. G. Lozano, MD Anderson) and Mdm2^+/+p53^−/− mice. All lines were confirmed mycoplasma-free (MycoSensor PCR Assay, Agilent #302109).

shRNA lentivirus generation and infection

Lentiviral shRNA constructs for MDM2 were provided by Dr. L. Mayo (IUSM) and purchased for TAp73 (TRCN6508, TRCN6511) and pLKO.1 non-targeting shRNA control (Millipore-Sigma; RRID:Addgene_8453). Lentivirus was produced by calcium phosphate transfection method into 293T cells (RRID:CVCL_0063). For infection of MDA-MB-231 and MDA-MB-436 cells, 1×10⁶ cells were placed into 10cm plates 16hr prior to infection. Following 16hr of exposure to lentiviral particles, cells were harvested and subsequently plated (considered time 0hr) for experimentation.

Cell survival and apoptosis analyses

Cells were placed in 96-well plates (2500–3000 cells/well, quadruplicate) and MTT (562nm; Millipore-Sigma #M2128) or MTS (DU4475 and MDA-MB-453 only; 492nm; Promega #G3580) survival/growth assays performed according to manufacturer’s protocol at 24hr intervals (kinetic analyses) or 48hr (dose responses) after adding compounds. Annexin-V positivity (Annexin-V Apoptosis Detection Kit; BD-Biosciences #556547), Caspase-3 activity (Caspase-3 Assay Kit; BD-Biosciences #556485), and Caspase-3/7 activity (Live Caspase-3/7 Green Detection Reagent; Invitrogen #C10723) were measured according to manufacturer’s instructions. Cell viability was determined by Trypan Blue dye (Gibco #15250061) exclusion. Propidium iodide (Millipore-Sigma #P4170) marked apoptotic subG1 DNA content, as we previously reported (77). FlowJo (BD-Biosciences; RRID:SCR_008520) was used for flow cytometric analyses.

Colony formation assays

Assays were performed as previously described (78). Briefly, MDA-MB-231 (250 cells/well) and MDA-MB-436 (300 cells/well) were placed, in triplicate, in 6-well plates. After 12 days in culture, colonies were stained with 0.5% crystal violet in methanol for 10min at room temperature and colonies (≥50 cells) were counted using a dissecting microscope. Representative pictures taken using an LG-G6 phone camera with 13MP standard-angle lens (no magnification).

Mammosphere assays

Mammospheres were established and maintained as previously described (79). Cells were placed in low-attachment 24-well plates (1,000 cells/well) for formation assays or 96-well plates (200 cells/well) for survival/ATP production assays (CellTiter-Glo 3D Cell Viability Assay, Promega #G9681) and Caspase-3/7 activity assays (Live Caspase-3/7 Green Detection Reagent, Invitrogen #C10723). IncuCyte Live-Cell Analysis System (Sartorius) was used for real-time quantitative live-cell imaging of Caspase-3/7 activity. Luminescence (survival/ATP production) and fluorescence (Caspase-3/7 activity) were measured and representative images captured using the Cytation-5 Cell Imaging Multimode Reader (BioTek). Mammosphere (≥50 cells) number and area were determined using the Gen5 image analysis tool on the Cytation-5.

Protein analysis

For Western blotting, whole-cell protein lysates were prepared as described (80) using ARF lysis buffer (50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA, 2.5mM EGTA, 0.1% Tween-20) with sonication (0.5 power level, two 7-second pulses separated by 2min on ice; VirTis VirSonic-600). For lysis comparisons, half of the cells were lysed using RIPA lysis buffer (50mM Tris pH 7.4, 150mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS). For immunoprecipitations, cells were lysed in EBC buffer (50mM Tris pH 7.5, 1mM EDTA, 1mM EGTA, 1% Triton X-100) using Dounce homogenizers as described (81). Antibodies are in Supplementary Table S2.

qRT-PCR

As we previously described (77), total RNA was isolated using TRIzol (Invitrogen #15596026) according to manufacturer’s instructions. SuperScript III First-strand Synthesis System (Invitrogen #18080051) generated cDNA prior to analysis of mRNA expression (triplicate) using RT²-SYBR Green ROX qPCR Mastermix (Qiagen #330521) on the StepOnePlus RT-PCR system (Applied Biosystems #4376600). Values were first normalized to β-ACTIN then made relative to vehicle or non-targeting shRNA controls and are presented as 2^−ΔΔCT. Primer sequences for human genes are in Supplementary Table S3; mouse primers were previously published (15).

RNA-sequencing and Hallmark gene signature analyses

MDA-MB-231 and MDA-MB-436 cells treated with MDM2-PROTAC or DMSO vehicle control (6μM, 16hr; triplicate) or expressing MDM2 shRNA or non-targeting shRNA control (48hr; triplicate) were harvested and RNA isolated (described above). Quality control assessment by Azenta/GENEWIZ included the Agilent TapeStation System and Qubit assay. Paired-end RNA-seq profiles of triplicate samples for each condition were generated from Illumina HiSeq-4000 and obtained from Azenta/GENEWIZ. RNA-seq data was analyzed as we previously described (82). Details are in Supplementary Methods. Differentially expressed genes (≥2 fold-change with Benjamini-Hochberg (83) adjusted P<0.05) were used to identify in which cancer Hallmark gene signatures they were significantly (FDR<0.05) enriched. We utilized WebGestalt (RRID:SCR_006786) (84) to conduct the enrichment analysis using the cancer Hallmark gene set available in the Molecular Signature DataBase (MSigDB; RRID:SCR_016863; (35)).

TAp73 ChIP

ChIP was performed as we previously reported (85), except DNA was sheared using a Q800R3 sonicator (Qsonica #Q800R3–110). ChIP products were quantified using RT²-SYBR Green ROX qPCR Mastermix (Qiagen #330521) on the StepOnePlus RT-PCR system (Applied Biosystems #4376600). Each immunoprecipitation was first normalized to the amount of DNA in the input then made relative to the appropriate IgG control. Antibody and primer information in Supplementary Tables S2 and S3, respectively.

Metabolic stability analysis

MDM2-PROTAC and controls (0.5μM) were incubated with 0.5mg/mL of mouse liver microsomes and an NADPH-regenerating system (cofactor solution) in potassium phosphate buffer (pH 7.4). Aliquots were taken at 0, 5, 15, 30, and 45min and reactions quenched with an acetonitrile solution containing an internal standard. Controls not containing the cofactor solution were also measured. Samples were analyzed by LC-MS/MS and results reported as peak area ratios of each analyte to internal standard. Intrinsic clearance (CL_int) and half-life (t_1/2) were determined from the first-order elimination constant by nonlinear regression. Alliance Pharma (Malvern, PA) conducted this study.

In vivo pharmacokinetic study

PK study (1-arm) in 18 CD-1 mice (n=3 mice/time point; RRID:IMSR_CRL:022) administered a single intraperitoneal injection of the MDM2-PROTAC at 10mg/kg dissolved in 10% DMSO, 10% solutol, and 80% PBS. Plasma samples were collected at 0.25, 0.5, 1, 2, 4, and 6hr following injection and analyzed by LC-MS/MS. Mean plasma concentrations of the MDM2-PROTAC were calculated using a linear regression analysis. Alliance Pharma (Malvern, PA) conducted this study.

Tumor xenografts

Mouse experiments were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee and followed all state and federal rules and regulations. For xenografts, 10×10⁶ MDA-MB-231 or MDA-MB-436 cells were injected (subcutaneous) into one flank of 6–8 week-old female athymic nude mice (Envigo; RRID:RGD_5508395). Mice were randomized (tumor size-matched) into treatment groups once tumor volumes reached 80mm³, and daily 50mg/kg intraperitoneal injections of MDM2-PROTAC, RG7112D, and vehicle control began and continued for 14 consecutive days for the survival studies or 3 days for the tumor response to treatment studies. Compounds were dissolved in 10% DMSO, 10% solutol, and 80% PBS. Tumors were measured using digital calipers and volumes calculated using the ellipsoid volume formula. A blood sample was collected and complete blood counts were determined (GENESIS Veterinary Hematology Analyzer, Oxford Science) 13 days after treatment began. Mice were euthanized once tumors reached 2000mm³. Tissues harvested were formalin-fixed, paraffin-embedded, sectioned, H&E stained, and histologically evaluated. For the tumor response to treatment studies, after 72hr of treatment, apoptotic analyses, as described above, of single-cell suspensions of tumors harvested from euthanized mice were performed.

Patient samples and patient derived explants

De-identified, fresh surgically-resected TNBC tumor and normal breast tissue from adjuvant-treated patients (Supplementary Table S1) were obtained with patient written informed consent (IRB protocol #20D.826) from the Thomas Jefferson University biorepository, which is a CAP certified lab that follows International Ethical Guidelines for Biomedical Research Involving Human Subjects. Patient-derived explants were established as previously reported (33,34). Following 48hr of treatment with the MDM2-PROTAC, control compound RG7112D, or DMSO vehicle control, explants were harvested and stained with H&E for histopathological review. A board-certified pathologist (Dr. Juan Palazzo) reviewed each blinded case for viable tumor and/or normal benign breast tissue. Immunohistochemistry for cleaved Caspase-3 (Supplementary Table S2) was performed on 4μm sections using the Vectorlab ABC-HRP detection Kit, HIER (pH 6.0) with Biocare Medical intelliPATH Autostainer. Blinded samples were scored for percent cleaved Caspase-3 positive cells by Dr. Palazzo and representative images taken. Mammosphere cultures from patient tumor samples were established, as described above. Following 72hr of treatment with the MDM2-PROTAC, RG7112, RG7112D, or DMSO vehicle control, mammospheres were evaluated for Caspase-3/7 activity and survival/ATP-production, as described above. Sequencing of TP53 cDNA was completed during or after experimentation, as published (86), except RNA was isolated using TRIzol and cDNA was generated, as described above. Primer sequences are in Supplementary Table S3.

Statistical analysis

All experiments were performed with a minimum of technical triplicates and at least two biological replicates per condition per cell line. Data are displayed as either mean ± standard deviation or mean ± standard error of the mean (indicated in the figure legend). For biological experiments, statistical significance was determined using unpaired, two-tailed Student t-tests when comparing two groups, 1-way ANOVA with Tukey multiple comparisons test when comparing more than two groups at one timepoint, 2-way ANOVA with Tukey multiple comparisons test when comparing more than two groups at multiple timepoints, or by log-rank test for survival analysis using GraphPad Prism software (RRID:SCR_002798). Longitudinal tumor growth analysis using 2-way ANOVA with Bonferroni correction, using the tool TumGrowth (87) determined significance of the difference in xenograft tumor volume overtime. To not distract from the data, a single P-value corresponding to the highest P-value in the figure panel (*P<X) is provided in the legend, unless otherwise noted; specific P-values are in the Supplementary spreadsheet. Spearman’s rank-correlation coefficients determined the association between MDM2-PROTAC-treated and MDM2 knockdown samples; the coefficient (ρ) and corresponding P-value are indicated in each plot. For RNA-seq data containing read counts, edgeR (RRID:SCR_012802) (88) was used to determine significance.

SIGNIFICANCE.

p53-inactivated TNBC is an aggressive, therapy resistant, and lethal breast cancer subtype. We designed a new compound targeting an unexpected vulnerability we identified in TNBC. Our MDM2-targeted degrader kills p53-inactivated TNBC cells, highlighting the requirement for MDM2 in TNBC cell survival and as a new therapeutic target for this disease.

Data availability

RNA-sequencing data sets are available on the Gene Expression Omnibus (RRID:SCR_005012) under accession number GSE214101.