p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell-cycle arrest or induction of apoptosis. However, the effects of p53 in antitumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell cross-talk, we studied murine syngeneic models treated with HDM201, a potent and selective second-generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells increased, including the CD103+ antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet+Eomes+ CD8+ T cells and the CD8+/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed in vitro and in vivo, which coincided with T-cell–mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, antitumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering antitumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors.

Significance:

This study provides a mechanistic rationale for combining checkpoint blockade immunotherapy with MDM2 inhibitors in patients with wild-type p53 tumors.

Tumor suppressor p53 is a potent transcription factor, which plays a central role in guarding genomic stability of the cell (1). Approximately 50% of all human cancers harbor p53 mutations, varying with tumor type ranging from ∼1% in papillary thyroid cancer to 95% in serous ovarian cancer (2, 3). In cancers where the TP53 gene is not mutated, the function of the p53 pathway is often suppressed through mechanisms that affect its stability and activity. One such mechanism is overexpression or deregulation of MDM2. MDM2, for which the human ortholog is known as HDM2, is an E3 ubiquitin ligase, which by direct binding negatively regulates p53 through ubiquitination and subsequent proteasomal degradation (4–6). HDM201 is a potent and selective small molecule inhibitor of the MDM2–p53 interaction (7, 8), which protects p53 from degradation, subsequently leading to its accumulation and activation, resulting in p53-dependent anti-proliferative effects and tumor growth inhibition (9) through transcriptional activation of downstream cell-cycle inhibitory genes (e.g., p21), and proapoptotic genes (e.g., PUMA and NOXA). It is the cooperative regulation of cell-cycle arrest genes versus apoptosis-inducing genes by p53 that ultimately determines cellular fate (10, 11). HDM201 inhibits the growth, in vitro and in vivo, of tumor models with functional wild-type (WT) p53 derived from a variety of cancer types (9).

Emerging evidence has also pointed to a role for p53 in immune modulation (12–15). Specifically, p53 has been implicated in induction of immunogenic cell death (13, 14), antigen processing and cytokine production (16, 17), immune checkpoint regulation, and immune tolerance (18, 19). However, the mechanism by which p53 affects immune function in the tumor microenvironment (TME) is still not well understood.

The programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) signaling axis is essential for maintaining immune homeostasis. PD-1 is expressed upon activation of lymphocytes (20). PD-1, upon interaction with its ligand PD-L1 or PD-L2, inhibits T-cell function, and although this is required to maintain peripheral tolerance (21), it can be detrimental to T-cell function during chronic viral infection and in tumor immunity. PD-1, along with other checkpoint receptors, is a marker of exhausted T cells in these chronic conditions (22, 23). PD-L1, a ligand of PD-1, which is expressed at very low level on macrophages and monocytes, can be induced on numerous cell types (including tumor-associated macrophages, T cells, and tumor cells) during inflammation and tumorigenesis (24–26). The expression of PD-L1 on tumor cells and its binding to PD-1 dampens T-cell activation and helps tumors to escape from detection and elimination by immune system (21, 27–29). Blockade of the interaction between PD-1 and PD-L1 can restore function in exhausted T cells, increase T-cell numbers in the TME and lead to an antitumor immune response preclinically, and in patients (30–32).

We investigated the contribution of tumor cells as well as immune cells to HDM201-induced tumor growth inhibition in Colon26 syngeneic tumors. We further explored the mechanism by which p53 affects the interaction of tumors cells and immune cells in the TME by characterizing syngeneic models treated with HDM201. HDM201 treatment led to increased expression of CD80 on Colon26 tumor cells, and the numbers of cross-presenting CD103+ dendritic cells (DC) infiltrating the tumor. It also increased a subset of CD8+ T cells that are Tbet+Eomes+, which have been reported to be more responsive to re-invigoration by PD-1 blockade (30, 33). HDM201 also increased the ratio of CD8+ T/Treg cells within the tumor, indicating a less immunosuppressive TME. Immunomodulatory effects of HDM201 in combination with blockade of PD-1/PD-L1 interaction significantly improved the antitumor response in preclinical syngeneic models with functional WT p53.

Reagents and cells

HDM201 was synthesized by Global Discovery Chemistry at Novartis. For in vitro studies, 10 mmol/L solutions were prepared in 100% DMSO. For in vivo experiments, HDM201 was dissolved in methylcellulose 0.5% w/V in 50 mmol/L phosphate buffer pH 6.8. An anti-PD-1 antibody (BioLegend, #135236, Clone: 29F.1A12) and anti-PD-L1 antibody (BioXCell, #BE0101, Clone: 10F.9G2) were used at 10 mg/kg.

Colon26 cells were obtained from Genomics Institute of the Novartis Foundation. p53 KO Colon26 cells were generated using CRISPR technology. 4T1 and Cloudman S91 were purchased from ATCC. MC38 cells were obtained from NCI, Mutz-3 from DSMZ, and LP6 from Dr. Christopher DM Fletcher at Brigham and Women's Hospital. Cells were authenticated by SNP analysis and tested negative for mycoplasma and virus infection by a PCR detection methodology (IDEXX BioAnalytics). Cells were used within 15 passages from thawing. Further details are provided in Supplementary Materials and Methods.

Animal studies

All animal studies were approved by the Novartis Institutes for BioMedical Research Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with the guidelines published in the Guide for the Care and Use of Laboratory Animals. All animals had access to food and water ad libitum. Eight-week-old female BALB/c and DBA-2 mice (The Jackson Laboratory), and C57BL/6 mice (Charles River Laboratories) were implanted subcutaneously on the right flank with either 2 × 105 Colon26 or 5 × 105 S91 or 1 × 105 MC38 cells, respectively. Tumor volume was measured with calipers twice per week, and when an average of 50 to 100 mm3 was reached, mice were randomized and orally administered vehicle or 40 mg/kg HDM201 every 3 hours for three times on day 1 and day 7. The anti-PD-1 or anti-PD-L1 antibody was administered at 10 mg/kg either by intraperitoneal injection or intravenously, on day 1, 3, 7, and 10.

Flow cytometric analysis

Tumors were collected on indicated days post-treatment and immediately processed. For flow cytometric analysis, cells were stained with Fixable Viability Dye-eFluor780 (eBioscience, #65-0865-14) or Live/Dead Fixable Yellow stain (Invitrogen, #L34959), followed by mouse Fc block (Miltenyi Biotec, #102949-182). The samples were stained with antibodies, listed in Supplementary Table S1. For intracellular staining, cells were fixed and permeabilized overnight using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, #00-5523-00), and stained with antibodies against intracellular proteins Foxp3 (Thermo Fisher Scientific, catalog no. 53-5773-82, RRID:AB_763537), T-bet (BioLegend, catalog no. 644815, RRID:AB_10896427), and Eomesodermin (Eomes; Life Technologies, #25-4875-82) for 1 hour. Data were acquired using an LSRFortessa (BD Biosciences) and analyzed using FlowJo (FlowJo, RRID:SCR_008520) software (Supplementary Materials and Methods). Gating strategies for T-cell populations and myeloid cell populations are depicted in Supplementary Figs. S1 and S2, respectively.

CD103+ bone marrow-derived DC differentiation

Bone marrow (BM) cells were isolated from femur/tibia of mice and cultured as described previously (34). Further details are provided in Supplementary Materials and Methods.

Mixed lymphocyte reaction

CD4 T cells from a healthy human donor were cocultured with the dendritic line, Mutz-3. Prior to coculture, Mutz-3 differentiation was induced using 100 ng/mL of GM-CSF (PeproTech), 80 ng/mL of IL4 (PeproTech), and low amounts of TNFα (2.5 ng/mL; PeproTech) for 5 days. Coculture supernatant was collected at day 3 for cytokine measurement by Sector Imager 2400 (MesoScale Discovery).

Characterization of tumor-antigen specific memory CD8+ T cells

Mice implanted with Colon26 tumors, with complete responses (CR) after HDM201 monotherapy or in combination with anti-PD-L1 or anti-PD-1 antibody treatments were sacrificed ∼100 days after tumor regression. Splenocytes were isolated and used to determine antitumor specific CD8+ T-cell memory by flow cytometry and ELISPOT assay. A Colon26-associated tumor antigen, AH1 (gp70423–431) peptide, which is a H-2Ld restricted, was used to measure tumor-antigen specific response (35, 36). For additional details, see Supplementary Materials and Methods.

In vitro PBMC coculture

Human tumor cell line LP6 was pretreated with either DMSO or 200 nmol/L HDM201 for 72 hours and then washed out. In parallel, fresh human PBMCs were isolated from a healthy donor and activated for 96 hours with human T-activator CD3/CD28 Dynabeads (Thermo Fisher Scientific, #111.31D) at a 1:1 bead to cell ratio. CD3/CD28 beads were then removed from the culture, and PBMCs added to the pretreated tumor cells at varying E:T (effector:tumor) ratios. Tumor cell number was monitored over time using an IncuCyte-ZOOM platform.

Statistical analysis

Survival was analyzed by Kaplan–Meier method and compared by log-rank test. Unpaired t test was used to make comparisons between two independent groups. One way ANOVA followed by Tukey test or Dunn test were used for comparisons among groups. Data are presented as mean ± SEM unless otherwise indicated. All data were analyzed using SigmaPlot (RRID:SCR_003210) and values of P < 0.05 were considered statistically significant.

HDM201 affects immune cell function and requires presence of the competent immune system for its antitumor activity

Consistent with its mechanism of action, HDM201 inhibits the growth of tumor cells with functional WT p53 (9). To evaluate whether HDM201 inhibits mouse tumor growth through tumor cell autonomous mechanisms or if its activity is also mediated by the TME, we tested the antitumor activity of HDM201 in Colon26, a WT p53 murine syngeneic tumor model grown in immunodeficient NSG or immunocompetent BALB/c mice. As shown in Fig. 1A and B and Supplementary Figs. S3A and S3B, antitumor effects with HDM201 were observed only when the Colon26 tumors were implanted into BALB/c mice. HDM201 failed to inhibit tumor growth when the tumor cells were implanted into NSG mice, despite the comparable induction of p53 target genes in both mouse strains (Fig. 1B; Supplementary Fig. S3C). These results suggest that the effect of HDM201 on Colon26 tumor cells alone in the absence of an intact immune system was not sufficient to inhibit tumor growth. This requirement of the competent immune system contrasts with observations made in human tumor models with MDM2 amplification, such as LP6 or SJSA-1, where HDM201 robustly inhibited the tumor growth in immunodeficient nude mice (9). However, tumors in which HDM201 is highly efficacious in nude mouse xenografts often display much lower GI50 values than Colon26 (Fig. 1C), suggesting that in cells exquisitely sensitive to HDM201 (e.g., LP6), a cell-autonomous therapeutic mechanism may predominate and is sufficient for antitumor effects.

Figure 1.

HDM201 modulates immune cell function and requires presence of a competent immune system for its antitumor activity. A, HDM201 inhibits Colon26 tumors grown in BALB/c mice, n = 10. **, P < 0.01 by one-way ANOVA followed by Tukey analysis. B, HDM201 fails to inhibit Colon26 tumor grown in NSG mice, n = 9. C, HDM201 dose–response curves for human or mouse syngeneic cell lines in vitro. D, Increases in percentage of CD103+CD11c+ DCs by HDM201 in vitro. Representative dot plots (left) and graph (right) of biological triplicates of two independent experiments. **, P < 0.01 by one-way ANOVA; ns, not significant. E, MDM2 inhibitors, HDM201 and RG7388, increase IL2 production in MLR assay. iDC and CD4+T cells were cocultured for 3 days. Data are presented as mean ± SD. *, P < 0.05 by Student t test.

Figure 1.

HDM201 modulates immune cell function and requires presence of a competent immune system for its antitumor activity. A, HDM201 inhibits Colon26 tumors grown in BALB/c mice, n = 10. **, P < 0.01 by one-way ANOVA followed by Tukey analysis. B, HDM201 fails to inhibit Colon26 tumor grown in NSG mice, n = 9. C, HDM201 dose–response curves for human or mouse syngeneic cell lines in vitro. D, Increases in percentage of CD103+CD11c+ DCs by HDM201 in vitro. Representative dot plots (left) and graph (right) of biological triplicates of two independent experiments. **, P < 0.01 by one-way ANOVA; ns, not significant. E, MDM2 inhibitors, HDM201 and RG7388, increase IL2 production in MLR assay. iDC and CD4+T cells were cocultured for 3 days. Data are presented as mean ± SD. *, P < 0.05 by Student t test.

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These initial data suggest that HDM201 may engage the immune system for its antitumor activity, but it is unclear how. Thus, we asked whether this could be through direct activation of T cells, or perhaps through effects on DCs. To determine whether HDM201 directly affects DCs, we used an ex vivo DC differentiation assay (37). After 9 days of culture in the presence of FLT3 ligand and GM-CSF, mouse bone marrow-derived precursor cells differentiated into CD103+ and CD103 DC subsets (Fig. 1D). HDM201 increased the number of DCs (Supplementary Fig. S3D) and the percentage of the CD103+CD11c+, a superior tumor antigen cross-presenting DC subset (Fig. 1D). These data indicate that HDM201 might affect DCs directly. To investigate whether HDM201 has effects on human immune cell function we utilized differentiated Mutz-3, a human DC cell line (38), in an ex vivo coculture assay (mixed lymphocyte reaction, MLR) to evaluate DC-mediated T-cell activation. Human T cells were cultured in presence and absence of Mutz-3 DCs and IL2 production was measured as a readout for T-cell proliferation. In the presence of DCs, IL2 production was significantly increased by HDM201 as well as by RG7388 (a chemically distinct MDM2 inhibitor; ref. 39), suggesting an MDM2/p53 pathway-dependent regulation of DC and T-cell function (Fig. 1E). Neither drug caused significant increase of IL2 by T cells alone or by DC cells alone (Fig. 1E). Taken together, these data indicated that HDM201 can directly enhance immune cell function ex vivo.

HDM201 modulates immune activity in TME in vivo

We next asked whether these direct effects of HDM201 on immune cells are the main drivers of observed antitumor efficacy. To test for effects of HDM201 intrinsic to cancer cells, we utilized CRISPR technology to delete p53 in the Colon26 model. Unlike in parental cells, p53 knockout (KO) clones were confirmed to have no detectable p53 protein or p21 upregulation in response to HDM201 treatment in vitro (Fig. 2A). KO cell lines grew unaffected in the presence of HDM201 in vitro and failed to modulate p53 target genes (Fig. 2B; Supplementary Figs. S4A and S4B). Among the tested p53 KO clones, clone 10 showed similar growth kinetics as parental Colon26 tumors (Supplementary Fig. S4C) and thus was chosen for comparison with the parental cells.

Figure 2.

HDM201 modulates immune activity in TME in vivo. A and B, Characterization of p53 knockout (p53KO) Colon26 clones in vitro, following 24-hour 1 μmol/L HDM201 treatment. Western blot analysis of p53 expression (A) and qPCR analysis of indicated target genes (B). C–K, Flow cytometry analysis of immune populations in vehicle (V) or HDM201 (H)-treated Colon26 parental and p53KO tumors. Five or 12 days post-treatment, tumors were collected and processed for flow analysis of the following populations: total immune cells (% CD45+) in total live cells (C); DC (% CD11c+MHCII+) in total CD45+ cells (D); %CD103+CD11c+ DC in total CD45+ (E); %CD103CD11c+DC in total CD45+ cells (F); granulocyticMDSC in total CD45+ cells (G); % of CD8+ T cells in total CD45+ cells (H); % of Tbet+Eomes+ cells in CD8+ T cells (I); %Treg (FoxP3+CD25+) in total CD4+ T cells (J); and ratio of CD8+T cells/Treg (K). Each point represents individual tumor/animal and lines represent mean ± SD, n = 7–10. *, P < 0.05; **, P < 0.01 by Student t test; ns, not significant.

Figure 2.

HDM201 modulates immune activity in TME in vivo. A and B, Characterization of p53 knockout (p53KO) Colon26 clones in vitro, following 24-hour 1 μmol/L HDM201 treatment. Western blot analysis of p53 expression (A) and qPCR analysis of indicated target genes (B). C–K, Flow cytometry analysis of immune populations in vehicle (V) or HDM201 (H)-treated Colon26 parental and p53KO tumors. Five or 12 days post-treatment, tumors were collected and processed for flow analysis of the following populations: total immune cells (% CD45+) in total live cells (C); DC (% CD11c+MHCII+) in total CD45+ cells (D); %CD103+CD11c+ DC in total CD45+ (E); %CD103CD11c+DC in total CD45+ cells (F); granulocyticMDSC in total CD45+ cells (G); % of CD8+ T cells in total CD45+ cells (H); % of Tbet+Eomes+ cells in CD8+ T cells (I); %Treg (FoxP3+CD25+) in total CD4+ T cells (J); and ratio of CD8+T cells/Treg (K). Each point represents individual tumor/animal and lines represent mean ± SD, n = 7–10. *, P < 0.05; **, P < 0.01 by Student t test; ns, not significant.

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We treated WT and KO p53 tumor-bearing BALB/c mice with HDM201 and collected tumors on day 5 and day 12 for phenotypic analysis of immune cells (gating strategies depicted in Supplementary Figs. S1 and S2). HDM201 treatment increased the percentage of CD45+ immune cells on day 5 in WT p53 tumors, but notably, no increase was observed in the p53 KO tumors, suggesting that p53 in cancer cells is required for increasing total immune cell infiltration (Fig. 2C). The percentage of total CD45+ cells in p53 KO was similar to WT tumors. In line with the absence of increased CD45+ cell infiltration in p53 KO tumors, no inhibition of tumor growth was seen in the efficacy studies (Supplementary Fig. S4C). Among the CD45+ cells, the percentage of DCs increased in WT and p53 KO tumors on day 5 (Fig. 2D). Similar to our in vitro findings (Fig. 1D), HDM201 significantly increased the CD103+ DC subset within the CD45+ cells (Fig. 2E). Notably, the percentage of CD103 DCs were also increased (Fig. 2F). In contrast to in vitro, gating for CD103+ and CD103 within CD11c+ DCs showed no specific enrichment of CD103+ DCs in the TME (Supplementary Figs. S4E and S4F). In line with previous reports for MDM2 inhibitors (40), Ly6G+ granulocytic-myeloid cell derived suppressor cells (G-MDSC) were reduced with HDM201 in WT tumors but not in p53 KO (Fig. 2G). HDM201 also decreased Ly6C+ monocytic (M)-MDSCs in WT tumors on day 12, whereas only a trend to lower percentages was detectable on day 5 (Supplementary Fig. S4G). No effects on F4/80+ macrophages were observed (Supplementary Fig. S4H).

Next, we characterized T-cell populations in the TME. In WT tumors, we detected an increase in CD8+ T cells with HDM201 treatment (Fig. 2H). In p53 KO tumors, no increase was observed on day 5. CD8+ T cells seemed to modestly increase in the KO tumors at the later time point, albeit only in 2 of 8 mice tested (i.e., day 12, Fig. 2H). These two mice displayed lower tumor volumes compared with others in the group (Supplementary Fig. S4D) and also appear to be outliers across several immunophenotypic markers analyzed (Fig. 2). T-bet and Eomesodermin (Eomes) are two T-Box transcription factors that play important roles in T-cell–mediated immune response against tumors. To determine whether HDM201 has an influence on T cells in tumors, the percentages of Tbet+Eomes+ T cells were examined. Within the CD8+ T-cell population, HDM201 treatment increased the percentage of Tbet+Eomes+ cells in WT but not in p53 KO tumors (Fig. 2I). Further analysis showed no significant changes in the Treg frequency (CD25+FOXp3+CD4+) in WT tumors, but in p53 KO tumors an increase in the Treg frequency was detectable on day 12 (Fig. 2J). Overall HDM201 treatment improved the CD8+ T cell/Treg ratio in WT tumors on Day 5, although minor trends toward improved CD8+ T cell/Treg ratios were also observed on day 12 in WT as well as in the two outlier p53 KO tumors (Fig. 2K).

In summary, we found that HDM201 promotes robust antitumor immunity that was associated with increased DC and T-cell responses in the TME. Importantly, our comparison of WT and p53 KO tumors suggests that tumor cell-mediated immunoregulatory mechanisms are key for antitumor immunity induced by HDM201.

HDM201 in combination with PD-1/PD-L1 blockade enhances antitumor responses in p53 WT tumors

A link between the tumor suppressor p53 and immune checkpoint regulators, including PD-1 and PD-L1 has been reported in cancer cells (19). In our studies, analysis of CD45 tumor cells revealed a statistically significant increase in PD-L1 expression in HDM201-treated Colon26 tumors, at both day 5 and day 12, which was dependent on the presence of WT p53 in tumor cells (Fig. 3A). The percent of PD-L1+ cells within CD45 population was not significantly changed (Supplementary Fig. S5A). In line with enhanced T-cell activation and potentially resulting T-cell exhaustion, HDM201 also caused increased frequencies of PD-1+ cells within T-cell populations, including CD8+ CTLs (Fig. 3B), CD4+ T cells, and Tregs (Supplementary Figs. S5B and S5C). Notably, increased frequencies of PD-1+ immune cells depended on WT p53 in tumor cells.

Figure 3.

HDM201 in combination with PD-1/PD-L1 blockade enhances antitumor responses in p53 WT tumors, but not in p53 mutant or KO tumors. A, Flow cytometry analysis of PD-L1 in CD45 cells in vehicle (V) or HDM201 (H)-treated Colon26 parental and p53KO tumors. Bar chart depicting median fluorescence intensity (MFI; left) and representative histograms of gated CD45 cells (right). B, Flow cytometry analysis of %PD-1+CD8+ cells in total CD45+ (left) or in total CD8+ T cells (middle) after vehicle (V) or HDM201 (H)-treated Colon26 parental and p53KO tumors. Representative flow plots (day 5) for PD-1 expression of CD8+ T cells (right). C–G, Effects of HDM201 in combination with anti-PD-1 antibody or anti-PD-L1 antibody on the survival of mice bearing Colon26 tumors (C and D), S91 tumors (E), MC38 tumors (F), and p53KO Colon26 tumors (G). The treatment with HDM201 is shown on the graph (arrows). Anti-PD-1 or anti-PD-L1 antibody was dosed at 10 mg/kg, twice a week for 2 weeks, n = 7–10 for each group. *, P < 0.05 by log-rank test; ns, not significant.

Figure 3.

HDM201 in combination with PD-1/PD-L1 blockade enhances antitumor responses in p53 WT tumors, but not in p53 mutant or KO tumors. A, Flow cytometry analysis of PD-L1 in CD45 cells in vehicle (V) or HDM201 (H)-treated Colon26 parental and p53KO tumors. Bar chart depicting median fluorescence intensity (MFI; left) and representative histograms of gated CD45 cells (right). B, Flow cytometry analysis of %PD-1+CD8+ cells in total CD45+ (left) or in total CD8+ T cells (middle) after vehicle (V) or HDM201 (H)-treated Colon26 parental and p53KO tumors. Representative flow plots (day 5) for PD-1 expression of CD8+ T cells (right). C–G, Effects of HDM201 in combination with anti-PD-1 antibody or anti-PD-L1 antibody on the survival of mice bearing Colon26 tumors (C and D), S91 tumors (E), MC38 tumors (F), and p53KO Colon26 tumors (G). The treatment with HDM201 is shown on the graph (arrows). Anti-PD-1 or anti-PD-L1 antibody was dosed at 10 mg/kg, twice a week for 2 weeks, n = 7–10 for each group. *, P < 0.05 by log-rank test; ns, not significant.

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We next asked whether blocking the PD-1/PD-L1 interaction would further enhance the antitumor effects of HDM201 in p53 WT syngeneic models. We tested HDM201 in combination with anti-PD-1 or anti-PD-L1 antibodies in the Colon26 tumor model. This model is not responsive to either anti-PD-1 or anti-PD-L1 antibody treatment as single agents (Fig. 3C and D). However, we observed a prominent increase in complete tumor regressions and long-term survival in their combination with HDM201 (Fig. 3C and D; Supplementary Figs. S5D–S5G). The median time to endpoint for vehicle control and anti-PD-1 antibody was 23 and 31.5 days for HDM201 as a monotherapy. The combination of HDM201 with anti-PD-1 antibody substantially prolonged the time to endpoint to 84 days (P < 0.05). Testing the HDM201 combination with an orthogonal checkpoint inhibitor, anti-PD-L1 antibody in the same model, resulted in similar antitumor activity (Fig. 3D). To confirm the combination benefit was applicable in different tumor models, we used an additional p53 WT syngeneic model, Cloudman S91 (S91), which is an NRAS WT melanoma model that harbors G506V BRAF mutation. HDM201 in combination with an anti-PD-L1 antibody increased the median time to endpoint from 39 days achieved with HDM201 monotherapy to 66 days (Fig. 3E; Supplementary Figs. S5H–S5K). Thus, we observed improved inhibition of tumor growth and overall survival benefit, however, the number of CRs resulting from anti-PD-L1 and HDM201 combination treatment of S91 tumors were lower compared with Colon26 tumors.

Next, we tested the requirement for functional p53, in the tumor cells, for anti-tumor activity with HDM201 as a single agent, and in combination with checkpoint blocking antibodies. We used the MC38 syngeneic model, which harbors a mutation in p53 and does not respond to HDM201 in vitro (Fig. 1C). As expected, treatment with HDM201 did not inhibit MC38 tumor growth as single agent. Likewise, the combination treatment with an anti-PD-L1 antibody did not show any improvement over anti-PD-L1 antibody effects alone (Fig. 3F). Because MC38 tumors have a very strong response to immune checkpoint blockade alone, we wanted to confirm that the lack of added benefit from HDM201 is not due to reasons beyond of p53 status. Thus, we also tested isogenic p53 KO Colon26 tumors, where similarly anti-PD-1 and HDM201 combination treatment did not induce any antitumor efficacy (Fig. 3G). Overall, these data demonstrate that a combination treatment regimen of HDM201 with PD-1/PD-L1 blockade significantly improves antitumor response and depends on p53 WT expression in tumor cells.

HDM201 promotes long-lasting tumor-specific immune memory responses

Given the immunomodulatory activity observed with HDM201 and its ability to combine with checkpoint blockade antibodies, we explored the durability and specificity of the resulting antitumor response. To test whether the antitumor response was antigen-specific, complete responder mice were rechallenged with a second injection of Colon26 cells on the opposite flank and subsequent injection of 4T1 cells into mammary fat pad (Fig. 4A). Mice that responded to HDM201 alone or to its combination with either anti-PD-L1 or anti-PD-1 did not develop tumors when rechallenged with Colon26 cells (Fig. 4B). When those same mice were however, re-implanted with 4T1 tumor cells, mice developed tumors (Fig. 4C). Comparatively, untreated naive mice developed tumors when implanted with either Colon26 or 4T1 (Fig. 4B and C). Rejection of tumor cells implanted at the distal site indicates development of systemic tumor-specific memory T-cell responses.

Figure 4.

HDM201 promotes development of durable tumor-specific memory T-cell responses. A, Representative treatment plan schematic to test long-term memory of mice that achieved CR. B, All mice that achieved CR after HDM201 alone (brown) or its combination with either anti-PD-L1 (green) or anti-PD-1 antibody (red) failed to develop tumors after the second contralateral implantation of Colon26 cells. Naive mice and CR mice were implanted with one million Colon26 cells on the left flank. C, All CR mice developed tumors after injection of 4T1 tumor cells into mammary fat pad. D, IFNγ production by splenocytes isolated from CR mice treated with HDM201 alone or in combination with anti-PD-1 antibody. M, media; IP, irrelevant peptide; AH1, AH1 peptide. E, Representative flow plots of two CR mice showing CD44 and AH-1/H-2Ld dextramer staining of CD8+ T cells. The irrelevant peptide (βGal) was used as a control. F, Bar graph showing frequency of CD44+ AH1+ cells within CD8+ T cells.

Figure 4.

HDM201 promotes development of durable tumor-specific memory T-cell responses. A, Representative treatment plan schematic to test long-term memory of mice that achieved CR. B, All mice that achieved CR after HDM201 alone (brown) or its combination with either anti-PD-L1 (green) or anti-PD-1 antibody (red) failed to develop tumors after the second contralateral implantation of Colon26 cells. Naive mice and CR mice were implanted with one million Colon26 cells on the left flank. C, All CR mice developed tumors after injection of 4T1 tumor cells into mammary fat pad. D, IFNγ production by splenocytes isolated from CR mice treated with HDM201 alone or in combination with anti-PD-1 antibody. M, media; IP, irrelevant peptide; AH1, AH1 peptide. E, Representative flow plots of two CR mice showing CD44 and AH-1/H-2Ld dextramer staining of CD8+ T cells. The irrelevant peptide (βGal) was used as a control. F, Bar graph showing frequency of CD44+ AH1+ cells within CD8+ T cells.

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To explore further whether HDM201 treatment induced antitumor memory T cell responses, splenocytes were isolated on day 100 post-tumor regression and stimulated in vitro with Colon26 associated antigen AH1 (gp70423–431) peptide assay (35, 41). Antigen-specific production of IFNγ by T cells was enumerated via ELISPOT and detected in all responders (Fig. 4D). Consistent with this, we observed an increase in frequency of CD44+AH1-specific CD8+ T cells in spleens of mice, which responded to HDM201, or the combination of HDM201 with anti-PD-1 antibody (Fig. 4E and F). Overall, these data demonstrated that treatment with HDM201 promoted the development of durable tumor-specific memory T-cell responses.

HDM201 induces tumor cell–specific gene expression changes in vitro and in vivo

The lack of any combination benefit between HDM201 and anti-PD-1 antibody in the isogenic p53 KO Colon26 cells suggest that the tumor cells themselves may play a role in influencing local immune cells when treated with HDM201. To investigate this possibility, we examined genome wide in vitro transcriptional changes caused by HDM201 over time, applying RNA-seq and looking for gene signatures known to modulate immune cell populations or impact sensitivity to immune checkpoint therapies (42). First, canonical changes in p53 target genes were confirmed (Fig. 5A). An expression fold-change heat map of immune checkpoint and costimulatory genes involved in immune cell activation in Fig. 5A reveals CD80 and GITR (TNFRSF18) as most upregulated by HDM201, persistent over 72 hours and consistent in both Colon26 and S91 cell lines. Importantly, this induction was not observed in the p53 KO cells (Fig. 5A). Additional costimulatory markers as well as additional gene signatures were evaluated, including the IFNγ-related mRNA profile reported to predict sensitivity to anti-PD-1 therapy (42), none of which showed clear and consistent modulation (Fig. 5B).

Figure 5.

Costimulatory molecules CD80 and GITR (Tnfrsf18) are upregulated in tumor cells in vitro. A and B, RNA-seq gene expression log2-fold change heat map, 1 μmol/L HDM201 normalized to DMSO for each time point (total of three DMSO and three HDM201 samples for each cell line). A, Immune checkpoint and costimulatory gene list (top); p53 signaling list (bottom). B, IFNγ-related transcriptional gene signature list. C–E, Flow cytometry analysis of CD80 and GITR cell surface expression after treatment with DMSO or 0.2, 0.5, or 1 μmol/L HDM201 for indicated time C, Representative histograms of CD80 in Colon26 parental (p53 WT) and p53KO cells (left), percentage of CD80 positive cells (bar graph, right). D and E, CD80 expression in S91 cells (D) and GITR expression in Colon26 and S91 cells (E). FACs data are representative of at least two independent experiments with biological duplicates.

Figure 5.

Costimulatory molecules CD80 and GITR (Tnfrsf18) are upregulated in tumor cells in vitro. A and B, RNA-seq gene expression log2-fold change heat map, 1 μmol/L HDM201 normalized to DMSO for each time point (total of three DMSO and three HDM201 samples for each cell line). A, Immune checkpoint and costimulatory gene list (top); p53 signaling list (bottom). B, IFNγ-related transcriptional gene signature list. C–E, Flow cytometry analysis of CD80 and GITR cell surface expression after treatment with DMSO or 0.2, 0.5, or 1 μmol/L HDM201 for indicated time C, Representative histograms of CD80 in Colon26 parental (p53 WT) and p53KO cells (left), percentage of CD80 positive cells (bar graph, right). D and E, CD80 expression in S91 cells (D) and GITR expression in Colon26 and S91 cells (E). FACs data are representative of at least two independent experiments with biological duplicates.

Close modal

CD80 and GITR are known to play important roles in T-cell activation. To confirm whether changes in mRNA translated to cell surface expression, we analyzed Colon26, S91, and p53KO Colon26 cells by flow cytometry (Fig. 5C). HDM201 treatment strongly augmented CD80 expression in a dose-dependent manner in both Colon26 and S91 cells (Fig. 5C and D; Supplementary Figs. S6A and S6B). Notably, in the isogenic p53 KO Colon26 cells, there was no change in CD80 cell surface expression (Fig. 5C). CD80 expression was remarkably lower in p53 KO cells at baseline compared with the parent cells, further supporting the notion that CD80 expression is linked to p53 (Fig. 5C). Despite the observed prominent increase in Gitr mRNA, the increase in the GITR surface protein expression was very small in both cell lines (Fig. 5E) when compared with the strong upregulation of CD80. The increase in frequency of GITR-positive cells and mean fluoresce intensity was also not consistent over time and drug doses in both cell lines (Fig. 5E; Supplementary Figs. S6A and S6B). In addition, we analyzed CD86 and PD-L1 expression by flow. No increase of CD86 was detected in Colon26 and S91 cells (Supplementary Figs. S6A–S6C). Surprisingly, cell surface expression of PD-L1 was downregulated in a dose-dependent manner (Supplementary Figs. S6A, S6B, and S6D).

Next, we examined whether HDM201 induces CD80 expression in tumors in vivo. We observed CD80 upregulation consistent with p53 and p21 upregulation in p53 WT Colon26 tumors, but not in p53 KO tumors (Fig. 6A). In addition, we found a significant increase in CD80 expression in CD45 population in the parental Colon26 tumors on both day 5 and day 12, whereas the induction of CD80 was completely absent in p53 KO tumors (Fig. 6B). Taken together these data demonstrate p53-dependent increase in costimulatory molecule CD80 in tumor cells treated with HDM201 both in vitro and in vivo.

Figure 6.

CD80 increase is observed in vivo on p53 WT tumors only and is associated with improved tumor cell killing by activated PBMCs in vitro. A, Western blot analysis of CD80, p21, and p53 in Colon26 parental and p53 KO tumors (in triplicates). B, Flow cytometry analysis of CD80 in CD45 cells in vehicle (V) or HDM201 (H)-treated Colon26 parental and p53KO tumors. Representative histograms of gated CD45 cells (left) and bar chart depicting median fluorescence intensity (MFI; right). Each point represents individual tumor/animal and lines represent mean ± SD (right), n = 7–10; *, P < 0.05; **, P < 0.01 by Student t test. ns, not significant. C, RT-PCR analysis of LP6 cells treated for 72 hours with 12.5, 50, or 200 nmol/L HDM201 in vitro (black bars), followed by drug washout and collection of RNA over an additional 72 hours (gray patterned bars). Fold change versus 72-hour DMSO sample was calculated. D, Coculture of LP6 cells pretreated with 200 nmol/L HDM201 and activated PBMCs from four healthy donors in vitro. Tumor cell number was monitored over indicated time and is expressed as percentage of cell number at T = 0 time point. Data are representative of at least two independent experiments, each performed with biological duplicates.

Figure 6.

CD80 increase is observed in vivo on p53 WT tumors only and is associated with improved tumor cell killing by activated PBMCs in vitro. A, Western blot analysis of CD80, p21, and p53 in Colon26 parental and p53 KO tumors (in triplicates). B, Flow cytometry analysis of CD80 in CD45 cells in vehicle (V) or HDM201 (H)-treated Colon26 parental and p53KO tumors. Representative histograms of gated CD45 cells (left) and bar chart depicting median fluorescence intensity (MFI; right). Each point represents individual tumor/animal and lines represent mean ± SD (right), n = 7–10; *, P < 0.05; **, P < 0.01 by Student t test. ns, not significant. C, RT-PCR analysis of LP6 cells treated for 72 hours with 12.5, 50, or 200 nmol/L HDM201 in vitro (black bars), followed by drug washout and collection of RNA over an additional 72 hours (gray patterned bars). Fold change versus 72-hour DMSO sample was calculated. D, Coculture of LP6 cells pretreated with 200 nmol/L HDM201 and activated PBMCs from four healthy donors in vitro. Tumor cell number was monitored over indicated time and is expressed as percentage of cell number at T = 0 time point. Data are representative of at least two independent experiments, each performed with biological duplicates.

Close modal

HDM201 treatment results in improved tumor cell killing by T cells

Next, we wanted to determine whether the increases in costimulatory CD80 molecules by tumor cells might have functional consequences on T-cell activation and their ability to kill tumor cells. To confirm that these effects are not limited to murine system, here we utilized LP6, a p53 WT human liposarcoma cell line. To exclude any direct effects of HDM201 on T-cell function, we pretreated tumor cells with HDM201 and removed the drug before coculture with T cells. To understand the durability of the HDM201-induced CD80 phenotype after drug removal, we pretreated cells for 72 hours with HDM201, removed the drug and followed the expression levels of CD80 and p53 target genes, p21 and MDM2, for an additional 72 hours by qRT-PCR. CD80 expression was upregulated up to 39-fold following HDM201 pretreatment, but then diminished over time returning to baseline levels approximately 48 hours following washout, in line with the decrease in p21 and Mdm2, which occurred by 24 hours (Fig. 6C). LP6 cells, pretreated with 200 nmol/L HDM201 and cocultured with activated human PBMCs, experienced higher amounts of tumor cell killing in cocultures compared with vehicle pretreated control (Fig. 6D). These effects were seen in the higher T-cell to cancer cell (E:T) ratios and reached its nadir around the 36 to 48 hours time points. This temporal effect coincides with the observed upregulation of CD80 expression and its decrease by 48 hours (Fig. 6C). These data overall suggest that the HDM201 driven increase of costimulatory CD80 molecules by tumor cells may promote T-cell–mediated cell killing and may provide a potential mechanism for an mdm2-p53–dependent tumor cell intrinsic enhancement of antitumor immunity.

The goal of this study was to investigate the immunomodulatory effects of MDM2/p53 signaling; to determine if observed immunomodulation by p53 was cell autonomous or if it occurred through interaction of tumor with the TME. Furthermore, we sought to test whether immunomodulatory effects of MDM2/p53 inhibitors can be exploited in combination with immune checkpoint blocking antibodies to provide additional antitumor benefit. Using syngeneic models, we demonstrated that HDM201 increased CD80 expression on tumor cells, showed enhancement of T-cell stimulation and an increase in the frequency of CD8+ T cells in the total CD45+ population. In addition, PD-L1 expression on CD45 cells and frequency of PD-1+ cells within T-cell populations were increased. As a result, blockade of PD-1/PD-L1 interaction enhanced HDM201 activity in p53 WT tumors but not in p53 mutant or p53 KO tumors.

Previous studies have reported that inflammation driven p53 activation in monocytes promotes their differentiation into active Ly6c+CD103+ monocytic antigen presenting cells within the tumor (43). In the TME, activation of p53 by HDM201 resulted in increased percentage of DCs, including CD103+ DCs, which are considered the most efficient cell type for tumor antigen cross-presentation and the priming of cytotoxic T lymphocytes (CTL). Because we also observed increased DCs in p53 KO tumors as well as in vitro culture systems, our data, in line with literature, support a role of p53 in DC differentiation, tumor antigen presentation and subsequent activation of CTLs. The exact mechanism of p53 induced increase of CD103+ DCs remains to be determined.

CD103+ DCs have the capacity to activate tumor-specific CD8+ T-cell cytotoxic responses. These cells actively transport tumor antigen to lymph nodes, which results in both direct CD8+ T-cell stimulation and antigen hand-off to resident myeloid cells within lymph nodes (44). This pathway has been demonstrated to facilitate the generation of tumor specific CD8+ T cells responses from lymph node resident naive CD8+ T cells (45). Our observation of an increase in frequency of Tbet+Eomes+CD8+ T cells suggests that HDM201 promotes the differentiation of Eomes high CD8+ T cells with potent cytotoxic activity, which are likely more exhausted (30, 33). Combination benefit of HDM201 with anti-PD-1 antibody/anti-PD-L1 antibody in p53 WT tumors is in agreement with this potential mechanism of action (46).

In addition to altering the TME, p53 activation has been shown to mediate chemotherapy or radiotherapy induced immunogenic cell death (47–49). Guo and colleagues previously reported that intra-tumor p53 activation by Nutlin-3, also an MDM2 inhibitor, increased components of damage-associated molecular patterns (13), which could make them more susceptible to killing by the immune system. Instead, we observed increased CD80 expression in Colon26 tumor cells in vitro and in vivo, which would suggest an alternative mechanism of MDM2/p53-mediated immune activation in tumors. This report also described that local intratumoral injection of Nutlin-3 triggered the immune response through p53-dependent immunogenic cell death, increased DC infiltration, and elimination of immunosuppressive MDSCs (13). We similarly observed an increase in DCs, a clear reduction of G-MDSCs and a slightly weaker reduction of M-MDSCs, which was however statistically significant at the later time point (i.e., day 12).

The combination benefit of HDM201 with anti-PD-1/PD-L1 antibody treatment was only observed in p53 WT tumors, indicating WT p53 is required for antitumor response. Utilizing isogenic p53 KO cells, we demonstrated that without intact p53, HDM201 fails to induce p53 responsive genes in vitro as well as in xenograft tumors in vivo. Furthermore, not only was the antitumor benefit of the single-agent HDM201 lost in p53 KO tumors, the strong antitumor effects of HDM201 in combination with anti-PD-1 antibody were also abolished in p53 KO tumors. Induction of T-cell costimulatory CD80 was also prevented in tumors with p53 knockout, overall suggesting that tumor p53 may play a direct role in influencing tumor immune microenvironment. Inactivation by a single-point mutation in p53 is also sufficient to prevent the p53-mediated response, as suggested by the lack of anti-tumor response in the MC38 model harboring p53-mutated protein.

In our genome wide studies, aside from Cd80, mRNA expression of Gitr was also increased. However, despite the observed prominent increase in Gitr mRNA, the increase in the GITR surface protein expression was very small in both cell lines tested. This discrepancy between mRNA and protein expression is not a rare observation, but in this case might require further investigation. Certainly if not significantly modulated on the cell surface, the likelihood of being functionally relevant for immune cell activation is uncertain. However, contribution of GITR to CD80 effects on local T-cell activation and adaptive antitumor immunity cannot be ruled out.

Notably, other costimulatory markers, such as CD86, as well as additional gene signatures known to regulate immune cell populations or impact sensitivity to immune checkpoint therapies were evaluated, but none showed clear and consistent modulation. Surprisingly, unlike in the in vivo setting, cell surface expression of PD-L1 was downregulated in vitro. One potential explanation for the difference might be that in vitro earlier time points were analyzed compared with in vivo, and the effect is delayed. A more likely reason however is that as opposed to in vitro, immune cells in the local TME in vivo may provide additional signals and enable an increase in PD-L1 expression.

Here presented data suggest that HDM201 may increase immune cell function through effects on DCs. In addition, comparison of WT and p53 KO tumors clearly demonstrates the requirement of MDM2/p53 signaling in tumor cells for effective antitumor immunity. Furthermore, HDM201 drives an increase of costimulatory CD80 molecules by tumor cells, implying that tumor cells themselves, may augment local T-cell activation and contribute to adaptive antitumor immunity. In support of this notion, our data show that HDM201 pretreatment of tumor cells indeed enhances PBMC-mediated tumor killing in vitro, which temporally coincides with increase of costimulatory CD80 molecule. We do recognize however that, the underlying mechanism might be more complex than sole CD80 upregulation.

Overall, our results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway. Thus, providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in cancer patients with WT p53. These preclinical observations are currently being tested in clinical trials, evaluating the combination of HDM201 with anti-PD-1 antibody in renal cell carcinoma and micro-satellite stable colorectal cancers (NCT02890069).

H.Q. Wang reports a patent for WO2019/180576 pending. J. Liang reports a patent for WO2019/180576 pending. C. Fabre reports other support from Novartis outside the submitted work. E. Halilovic reports a patent for WO2019/180576 pending. No disclosures were reported by the other authors.

H.Q. Wang: Data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–review and editing. I.J. Mulford: Data curation, software, formal analysis, validation, investigation, visualization, methodology, writing–review and editing. F. Sharp: Writing–original draft. J. Liang: Data curation, investigation, methodology. S. Kurtulus: Data curation, supervision, writing–original draft. G. Trabucco: Data curation, investigation, methodology. D.S. Quinn: Data curation, software, investigation, visualization, methodology, writing–review and editing. T.A. Longmire: Data curation, investigation. N. Patel: Data curation, investigation, visualization, methodology. R. Patil: Data curation, investigation, visualization, methodology. M.D. Shirley: Data curation, software. Y. Chen: Data curation. H. Wang: Data curation. D.A. Ruddy: Supervision. C. Fabre: Writing–review and editing. J.A. Williams: Supervision. P.S. Hammerman: Supervision, writing–review and editing. J. Mataraza: Supervision, writing–review and editing. B. Platzer: Data curation, formal analysis, supervision, validation, visualization, methodology, writing–original draft, project administration, writing–review and editing. E. Halilovic: Conceptualization, data curation, formal analysis, supervision, validation, visualization, methodology, writing–original draft, project administration, writing–review and editing.

This work was funded in full by Novartis Institutes for BioMedical Research.

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

1.
Lane
DP
. 
Cancer. p53, guardian of the genome
.
Nature
1992
;
358
:
15
6
.
2.
Bouaoun
L
,
Sonkin
D
,
Ardin
M
,
Hollstein
M
,
Byrnes
G
,
Zavadil
J
, et al
TP53 variations in human cancers: new lessons from the IARC TP53 database and genomics data
.
Hum Mutat
2016
;
37
:
865
76
.
3.
Pfister
NT
,
Prives
C
. 
Transcriptional regulation by wild-type and cancer-related mutant forms of p53
.
Cold Spring Harb Perspect Med
2017
;
7
:
a026054
.
4.
Honda
R
,
Tanaka
H
,
Yasuda
H
. 
Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53
.
FEBS Lett
1997
;
420
:
25
7
.
5.
Kussie
PH
,
Gorina
S
,
Marechal
V
,
Elenbaas
B
,
Moreau
J
,
Levine
AJ
, et al
Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain
.
Science
1996
;
274
:
948
53
.
6.
Haupt
Y
,
Maya
R
,
Kazaz
A
,
Oren
M
. 
Mdm2 promotes the rapid degradation of p53
.
Nature
1997
;
387
:
296
9
.
7.
Furet
P
,
Masuya
K
,
Kallen
J
,
Stachyra-Valat
T
,
Ruetz
S
,
Guagnano
V
, et al
Discovery of a novel class of highly potent inhibitors of the p53-MDM2 interaction by structure-based design starting from a conformational argument
.
Bioorg Med Chem Lett
2016
;
26
:
4837
41
.
8.
Holzer
P
. 
Discovery of potent and selective p53-MDM2 protein-protein interaction inhibitors as anticancer drugs
.
Chimia
2017
;
71
:
716
21
.
9.
Jeay
S
,
Ferretti
S
,
Holzer
P
,
Fuchs
J
,
Chapeau
EA
,
Wartmann
M
, et al
Dose and schedule determine distinct molecular mechanisms underlying the efficacy of the p53-MDM2 inhibitor HDM201
.
Cancer Res
2018
;
78
:
6257
67
.
10.
Kruse
JP
,
Gu
W
. 
Modes of p53 regulation
.
Cell
2009
;
137
:
609
22
.
11.
Schlereth
K
,
Charles
JP
,
Bretz
AC
,
Stiewe
T
. 
Life or death: p53-induced apoptosis requires DNA binding cooperativity
.
Cell Cycle
2010
;
9
:
4068
76
.
12.
Guo
G
,
Cui
Y
. 
New perspective on targeting the tumor suppressor p53 pathway in the tumor microenvironment to enhance the efficacy of immunotherapy
.
J Immunother Cancer
2015
;
3
:
9
.
13.
Guo
G
,
Yu
M
,
Xiao
W
,
Celis
E
,
Cui
Y
. 
Local activation of p53 in the tumor microenvironment overcomes immune suppression and enhances antitumor immunity
.
Cancer Res
2017
;
77
:
2292
305
.
14.
Moore
EC
,
Sun
L
,
Clavijo
PE
,
Friedman
J
,
Harford
JB
,
Saleh
AD
, et al
Nanocomplex-based TP53 gene therapy promotes anti-tumor immunity through TP53- and STING-dependent mechanisms
.
Oncoimmunology
2018
;
7
:
e1404216
.
15.
Munoz-Fontela
C
,
Mandinova
A
,
Aaronson
SA
,
Lee
SW
. 
Emerging roles of p53 and other tumour-suppressor genes in immune regulation
.
Nat Rev Immunol
2016
;
16
:
741
50
.
16.
Slatter
TL
,
Wilson
M
,
Tang
C
,
Campbell
HG
,
Ward
VK
,
Young
VL
, et al
Antitumor cytotoxicity induced by bone-marrow-derived antigen-presenting cells is facilitated by the tumor suppressor protein p53 via regulation of IL-12
.
Oncoimmunology
2016
;
5
:
e1112941
.
17.
Zhu
K
,
Wang
J
,
Zhu
J
,
Jiang
J
,
Shou
J
,
Chen
X
. 
p53 induces TAP1 and enhances the transport of MHC class I peptides
.
Oncogene
1999
;
18
:
7740
7
.
18.
Cortez
MA
,
Ivan
C
,
Valdecanas
D
,
Wang
X
,
Peltier
HJ
,
Ye
Y
, et al
PDL1 Regulation by p53 via miR-34
.
J Natl Cancer Inst
2016
;
108
:
djv303
.
19.
Yoon
KW
,
Byun
S
,
Kwon
E
,
Hwang
SY
,
Chu
K
,
Hiraki
M
, et al
Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53
.
Science
2015
;
349
:
1261669
.
20.
Agata
Y
,
Kawasaki
A
,
Nishimura
H
,
Ishida
Y
,
Tsubata
T
,
Yagita
H
, et al
Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes
.
Int Immunol
1996
;
8
:
765
72
.
21.
Keir
ME
,
Liang
SC
,
Guleria
I
,
Latchman
YE
,
Qipo
A
,
Albacker
LA
, et al
Tissue expression of PD-L1 mediates peripheral T cell tolerance
.
J Exp Med
2006
;
203
:
883
95
.
22.
Barber
DL
,
Wherry
EJ
,
Masopust
D
,
Zhu
B
,
Allison
JP
,
Sharpe
AH
, et al
Restoring function in exhausted CD8 T cells during chronic viral infection
.
Nature
2006
;
439
:
682
7
.
23.
Wherry
EJ
. 
T cell exhaustion
.
Nat Immunol
2011
;
12
:
492
9
.
24.
Keir
ME
,
Butte
MJ
,
Freeman
GJ
,
Sharpe
AH
. 
PD-1 and its ligands in tolerance and immunity
.
Annu Rev Immunol
2008
;
26
:
677
704
.
25.
Gibbons Johnson
RM
,
Dong
H
. 
Functional expression of programmed death-ligand 1 (B7-H1) by immune cells and tumor cells
.
Front Immunol
2017
;
8
:
961
.
26.
Shen
X
,
Zhang
L
,
Li
J
,
Li
Y
,
Wang
Y
,
Xu
ZX
. 
Recent findings in the regulation of programmed death ligand 1 expression
.
Front Immunol
2019
;
10
:
1337
.
27.
Iwai
Y
,
Ishida
M
,
Tanaka
Y
,
Okazaki
T
,
Honjo
T
,
Minato
N
. 
Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by PD-L1 blockade
.
Proc Natl Acad Sci U S A
2002
;
99
:
12293
7
.
28.
Juneja
VR
,
McGuire
KA
,
Manguso
RT
,
LaFleur
MW
,
Collins
N
,
Haining
WN
, et al
PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity
.
J Exp Med
2017
;
214
:
895
904
.
29.
Lau
J
,
Cheung
J
,
Navarro
A
,
Lianoglou
S
,
Haley
B
,
Totpal
K
, et al
Tumour and host cell PD-L1 is required to mediate suppression of anti-tumour immunity in mice
.
Nat Commun
2017
;
8
:
14572
.
30.
Blackburn
SD
,
Shin
H
,
Freeman
GJ
,
Wherry
EJ
. 
Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade
.
Proc Natl Acad Sci U S A
2008
;
105
:
15016
21
.
31.
Mkrtichyan
M
,
Najjar
YG
,
Raulfs
EC
,
Abdalla
MY
,
Samara
R
,
Rotem-Yehudar
R
, et al
Anti-PD-1 synergizes with cyclophosphamide to induce potent anti-tumor vaccine effects through novel mechanisms
.
Eur J Immunol
2011
;
41
:
2977
86
.
32.
Rosenblatt
J
,
Glotzbecker
B
,
Mills
H
,
Vasir
B
,
Tzachanis
D
,
Levine
JD
, et al
PD-1 blockade by CT-011, anti-PD-1 antibody, enhances ex vivo T-cell responses to autologous dendritic cell/myeloma fusion vaccine
.
J Immunother
2011
;
34
:
409
18
.
33.
Paley
MA
,
Kroy
DC
,
Odorizzi
PM
,
Johnnidis
JB
,
Dolfi
DV
,
Barnett
BE
, et al
Progenitor and terminal subsets of CD8+ T cells cooperate to contain chronic viral infection
.
Science
2012
;
338
:
1220
5
.
34.
Mayer
CT
,
Ghorbani
P
,
Nandan
A
,
Dudek
M
,
Arnold-Schrauf
C
,
Hesse
C
, et al
Selective and efficient generation of functional Batf3-dependent CD103+ dendritic cells from mouse bone marrow
.
Blood
2014
;
124
:
3081
91
.
35.
Huang
AY
,
Gulden
PH
,
Woods
AS
,
Thomas
MC
,
Tong
CD
,
Wang
W
, et al
The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product
.
Proc Natl Acad Sci U S A
1996
;
93
:
9730
5
.
36.
Rice
J
,
Buchan
S
,
Stevenson
FK
. 
Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen
.
J Immunol
2002
;
169
:
3908
13
.
37.
Mayer
ML
,
Phillips
CM
,
Stadnyk
AW
,
Halperin
SA
,
Lee
SF
. 
Synergistic BM-DC activation and immune induction by the oral vaccine vector Streptococcus gordonii and exogenous tumor necrosis factor
.
Mol Immunol
2009
;
46
:
1883
91
.
38.
Masterson
AJ
,
Sombroek
CC
,
De Gruijl
TD
,
Graus
YM
,
van der Vliet
HJ
,
Lougheed
SM
, et al
MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+ precursors
.
Blood
2002
;
100
:
701
3
.
39.
Ding
Q
,
Zhang
Z
,
Liu
JJ
,
Jiang
N
,
Zhang
J
,
Ross
TM
, et al
Discovery of RG7388, a potent and selective p53-MDM2 inhibitor in clinical development
.
J Med Chem
2013
;
56
:
5979
83
.
40.
Panka
DJ
,
Liu
Q
,
Geissler
AK
,
Mier
JW
. 
Effects of HDM2 antagonism on sunitinib resistance, p53 activation, SDF-1 induction, and tumor infiltration by CD11b+/Gr-1+ myeloid derived suppressor cells
.
Mol Cancer
2013
;
12
:
17
.
41.
Hamaguchi
M
,
Eto
M
,
Kamiryo
Y
,
Takeuchi
A
,
Harano
M
,
Tatsugami
K
, et al
Allogeneic cell therapy from immunized donors with tumor antigen peptide enhances the antitumor effect after cyclophosphamide-using non-myeloablative allogeneic hematopoietic cell transplantation
.
Cancer Sci
2009
;
100
:
138
43
.
42.
Ayers
M
,
Lunceford
J
,
Nebozhyn
M
,
Murphy
E
,
Loboda
A
,
Kaufman
DR
, et al
IFN-gamma-related mRNA profile predicts clinical response to PD-1 blockade
.
J Clin Invest
2017
;
127
:
2930
40
.
43.
Sharma
MD
,
Rodriguez
PC
,
Koehn
BH
,
Baban
B
,
Cui
Y
,
Guo
G
, et al
Activation of p53 in immature myeloid precursor cells controls differentiation into Ly6c(+)CD103(+) monocytic antigen-presenting cells in tumors
.
Immunity
2018
;
48
:
91
106
.
44.
Ruhland
MK
,
Roberts
EW
,
Cai
E
,
Mujal
AM
,
Marchuk
K
,
Beppler
C
, et al
Visualizing synaptic transfer of tumor antigens among dendritic cells
.
Cancer Cell
2020
;
37
:
786
99
.
45.
Roberts
EW
,
Broz
ML
,
Binnewies
M
,
Headley
MB
,
Nelson
AE
,
Wolf
DM
, et al
Critical role for CD103(+)/CD141(+) dendritic cells bearing CCR7 for tumor antigen trafficking and priming of T cell immunity in melanoma
.
Cancer Cell
2016
;
30
:
324
36
.
46.
Fang
DD
,
Tang
Q
,
Kong
Y
,
Wang
Q
,
Gu
J
,
Fang
X
, et al
MDM2 inhibitor APG-115 synergizes with PD-1 blockade through enhancing antitumor immunity in the tumor microenvironment
.
J Immunother Cancer
2019
;
7
:
327
.
47.
Kroemer
G
,
Galluzzi
L
,
Zitvogel
L
. 
Immunological effects of chemotherapy in spontaneous breast cancers
.
Oncoimmunology
2013
;
2
:
e27158
.
48.
Galluzzi
L
,
Buque
A
,
Kepp
O
,
Zitvogel
L
,
Kroemer
G
. 
Immunological effects of conventional chemotherapy and targeted anticancer agents
.
Cancer Cell
2015
;
28
:
690
714
.
49.
Zitvogel
L
,
Galluzzi
L
,
Smyth
MJ
,
Kroemer
G
. 
Mechanism of action of conventional and targeted anticancer therapies: reinstating immunosurveillance
.
Immunity
2013
;
39
:
74
88
.