P53 Missense Mutations Research Articles

Abstract LB303: Suppression of stress granule formation is a novel vulnerability imposed by mutant p53

Abstract Mutation of the tumor suppressor p53 is the most common cancer specific event. Most of these mutations are missense mutations in the DNA-binding domain of p53, causing loss of wild-type p53 (wtp53) transcriptional activity. Missense mutant p53 (mutp53) also enhances cancer malignancy in a manner independent of wtp53. Hence, mutp53 is an ideal therapeutic target due to its uniqueness and high expression levels in cancers; however, directly targeting mutp53 protein itself has been challenging. To better understand the mechanism of oncogenic function of mutp53, we performed in silico TCGA database analysis and found that patients with missense mutp53-carrying hepatocellular carcinoma (HCC), but not other types of cancer, had unexpectedly better overall survival than patients with p53-null HCC. Since sorafenib (SOR) has historically been used as a single agent chemotherapy for HCC, unlike other cancer types, we hypothesized that SOR treatment may have yet unappreciated advantages for treating mutp53-expressing tumors. Indeed, knockdown of mutp53 rendered HCC cells and xenografts more resistant to SOR. Consistent with a report showing that SOR induces ER stress-mediated pro-survival stress granule (SG) formation, mutp53-expressing HCC cells, treated with SOR or other ER stress inducers, formed significantly fewer SGs with increased apoptosis, as compared with mutp53-knockdown cells. This result was partly due to mutp53’s ability to interact with an eIF2α kinase PERK and a key SG core protein, G3BP1. Moreover, the oligomerization domain of mutp53 was required for the interaction of mutp53 with G3BP1 and subsequent suppression of ER-stress-mediated SG formation and survival. These results suggest that inhibition of SG formation is a novel vulnerability imposed by mutp53, leading to sensitization of cancer cells to ER stress. Our results also emphasize the use of ER stress inducers to treat mutp53-expressing tumors and mutp53 as a biomarker of SOR sensitivity. Citation Format: Elizabeth Thoenen, Atul Ranjan, Alejandro Parrales, Shigeto Nishikawa, Dan Dixon, Sugako Oka, Tomoo Iwakuma. Suppression of stress granule formation is a novel vulnerability imposed by mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB303.

Abstract 640: In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations

Abstract p53 tumor suppressor mutations result in loss of function of p53 in approximately 50% of all human cancers.TP53 missense, nonsense mutations and p53 deletion are present in about 50%, 12% and 5% of all p53 driven cancers, respectively. To date there is no effective treatment for rescuing p53 function in cancers using a pan-p53 approach. The use of ADGN-531 is a new potent strategy combining mRNA with a tumor selective peptide nanocarrier to rescue of p53 tumor suppressor function. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides in the form of nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring various p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and NSCLC NCI-H358 (p53 null/KRASG12C) mouse xenografts at a dose of 0.25-1.0 mg/kg weekly. ADGN-531/sotorasib synergy was evaluated in vitro on NCI-H358 (p53 null/KRASG12C) and Mia-PACA (p53R48W/KRASG12C) cells and in vivo on NCI-H358 mouse xenograft. ADGN-531 markedly inhibits the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations or p53-deletion, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition in SaOs2, SW403 and H358 xenografts independent of KRAS status. We demonstrated a strong synergy in a NSCLC H358 xenograft (tumor regression 80%) when restoring P53 function with ADGN-531 (0.5mg/kg, weekly) along with inhibition of KRASG12C mutant using sotorasib (10mg/kg, daily). ADGN-531 potentiated the effect of sotorasib on sotorasib resistant cells harboring high level of KRASG12C in permanent active state. ADGN-531 treatments are well tolerated, no sign of clinical toxicity was detected after single or repeated administrations. ADGN-531 is effective in rescuing p53 function both in vitro and in vivo across a wide range of p53 alterations. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy in P53 altered tumors independent of other driver mutations e.g., KRAS, or with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Veronique Josserand, Melanie Guidetti, Neil Desai. In vivo rescue of p53 tumor suppressor function with ADGN-531 across Pan-p53 alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 640.

Abstract 631: Functional genetics of APR-246 rescuable TP53 mutants

Abstract TP53, often referred to as the guardian of the genome, encodes for the p53 tumor suppressor protein, and is one of the most frequently mutated genes in human cancers. Some cancer sub-types, such as non-small cell lung carcinoma (NSCLC) and high grade serous carcinoma (HGSC), have high TP53 mutation rates reaching up to 56% and 96%, respectively. Given its multi-faceted role in human cancers, there have been several emerging therapeutic approaches targeting mutant p53. APR-246 is a small molecule drug currently in clinical trials for the functional rescue of p53 missense mutants, the most common type of p53 mutant protein, across various cancer types. APR-246 is proposed to bind mutant p53 and promote thermodynamic stabilization of the protein allowing it to regain wild-type-like conformation and consequentially regain WT-like functionality. However, there has yet to be a functional genomic screening of p53 mutants to systematically catalog which mutants are rescuable by APR-246. To address this critical gap, we have performed functional genetic screenings using a TP53 mutagenesis library. This pooled lentiviral library contains ~8,000 amino acid variants, spanning the entirety of the TP53 gene. Following stable transduction of this library into p53-null NSCLC and HGSOC cell lines, we performed functional screenings against sublethal doses of APR-246. Next-generation sequencing of the untreated and treated cell population allows for comparison of variant representation and determination of APR-246-sensitivity of p53 mutants. Preliminary statistical analysis of our screens has identified a pool of 117 “hit” mutants which are significantly depleted following treatment, indicative of APR-246-sensitivity. Two of these mutants, C242F and G266V, are non-functional, damaging mutations which occur in patients. Identifying p53 mutants that can be rescued by APR-246 will help identify which patients may benefit most from treatment with the drug. Citation Format: Anais Saunders, Caili Tong, Anthony N. Karnezis, Gary S. Leiserowitz, Jeremy R. Chein. Functional genetics of APR-246 rescuable TP53 mutants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 631.

Abstract 7275: GS-P-328, a brain-penetrant small molecule p53 Y220C reactivator for tumors harboring p53 Y220C mutation

Abstract As a tumor suppressor, p53 can prevent tumor development through multiple pathways. However, mutation of TP53 and the resultant inactivation of p53 allow evasion of tumor cell death and rapid tumor progression. The oncogenic Y220C mutation is the ninth most frequent p53 missense mutation in cancer. Y220C mutation creates a narrow, hydrophobic pocket on the surface of the p53 DBD that reduces its thermal stability. As a result, the Y220C mutation causes a rapidly unfold of the p53 protein under physiological conditions, abrogates p53 signaling and drives tumorigenesis. There are approximately 100,000 new Y220C mutation cancer cases per year worldwide with limited treatment choices. The initial clinical evaluation of PC14586 that can restore missense-mutant p53 protein has seen efficacy in patients harboring p53 Y220C mutation even though there is still room for improvement. Here we report a brain-penetrable, highly potent p53 Y220C reactivator which has great in vitro activity as well as induce tumor shrinkage in multiple tumor models at a much lower systematic exposure comparing to PC14586. In vitro, GS-P-328 could bind to p53 Y220C protein with a single nM potency and potently induce cell death in a wide range of p53 Y220C tumor cells while have no anti-proliferative effects with p53 WT cells. GS-P-328 is also active in patient-derived cell (PDC) models in various tumor background including SCLC, gastric, ovarian and colon. In vivo, GS-P-328, at approximately 1/3 of systematic exposure comparing to a leading clinical reactivator, causes tumor shrinkage in multiple CDX models. GS-P-328 could induce MDM2/p21 upregulation in cell systems. In vivo, GS-P-328 could induce 2 fold and more increase in MDM2/p21 protein levels in tumor tissues at approximately 1/3 of plasma concentrations comparing to PC14586. More importantly, in a NUGC3-luc intracranial model, GS-P-328 could strongly inhibit growth of cancer cells in the mouse brain. GS-P-328 has a favorable ADME profile in rodents, dogs and NHPs, as well as a much better safety profile. GS-P-328 is now under IND-enabling study and P1 study is planned in early 2025. Citation Format: Meng Liu, Kaijun Geng, Biao Lu, Yuanfeng Xia, Fanglong Yang. GS-P-328, a brain-penetrant small molecule p53 Y220C reactivator for tumors harboring p53 Y220C mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7275.

Abstract B087: Mutant-p53 amplifies Cxcl1 expression from distal enhancers blunting immune checkpoint inhibition efficacy in pancreatic cancer

Abstract Pancreatic ductal Adenocarcinoma (PDAC) is an aggressive malignancy complicated by poor early diagnosis and a lack of response to traditional treatments. It is characterized by a desmoplastic stroma, a lack of infiltration and activation of T cells, and a low mutational burden. The genetic landscape of PDAC is defined by activating KRAS mutations (~90%) and p53 alterations (~70%), but the molecular switches perturbed by these genetic aberrations remain unclear. p53 missense mutations, unlike mutations resulting in the loss of p53, are considered to acquire tumor-supporting functions. But these novel functions remain uncharacterized. The ambiguity in the molecular mechanism of mutant-p53 is further exacerbated by the existence of various types of p53 mutations. The majority of p53 mutations are missense mutations in the DNA binding domain. The repertoire of transcription factors (TFs) it can interact with and the vast regulatory landscape of each TF—composed of gene promoters and distal enhancers—present obstacles in understanding the molecular mechanisms promoting PDAC. In this study, we examined how a common p53 missense mutation in PDAC plays a role in weakening the Immune checkpoint Inhibitors (ICIs) efficacy. Using cells derived from a genetically engineered mouse model (GEMM) of PDAC with activating KRAS mutation (KrasG12D/+) and a p53 missense mutation (p53R172H/-), we found that the PDAC tumorigenesis and resistance to ICIs are dependent on the mutant-p53. We used isogenic p53-null PDAC cells and the restoration of p53R172H in p53-null cells to demonstrate the role of p53R172H in controlling the expression of immunosuppressive chemokine genes such as Cxcl1. p53R172H deletion attenuated PDAC tumor growth, increased the influx of cytotoxic T-cells, and sensitized the tumor to ICIs. The p53R172H-mediated TME reprogramming was replicated by the deletion of the Cxcl1 gene, suggesting the anti-tumorigenic effect of p53R172H was mediated by the Cxcl1 gene. We probed the mechanism of Cxcl1 expression dependence on p53R172H. We found that in conjunction with NF-kB, p53R172H occupies the distal transcription regulatory elements (dTREs) of the Cxcl1 gene harboring NF-kB binding sites. Strikingly, deletion of the Cxcl1 dTREs in PDAC cells recapitulates the phenotypes of p53R172H deletion and Cxcl1 deletion in terms of tumor size, immune landscape of the TME, and ICI responsiveness. Furthermore, we examined the interplay between p53R172H and NF-kB and found that the p53R172H physically interacts with the NF-kB subunit RelA and facilitates its nuclear translocation. Overall, we characterize how a common p53 mutation in PDAC co-opts non-coding regulatory DNA to augment the expression of selective chemokine genes and establishes an immunosuppressive TME to shield the therapeutic benefits of ICIs. Citation Format: Dig B. Mahat, Heena Kumra, Emily Metcalf, Sarah Castro, Kim Nguyen1, Arundeep Singh, William W. Ho, Ivy Chen, Brandon Sullivan, Leon Yim, Enrico Moiso, Vikash Chauhan, Hernandez Moura Silva, Stefani Spranger, Rakesh Jain, Phillip A. Sharp. Mutant-p53 amplifies Cxcl1 expression from distal enhancers blunting immune checkpoint inhibition efficacy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B087.

P53 deficient breast cancer cells reprogram preadipocytes toward tumor-protective immunomodulatory cells.

The TP53 gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients.

Pharmacological reactivation of p53 in the era of precision anticancer medicine.

p53, which is encoded by the most frequently mutated gene in cancer, TP53, is an attractive target for novel cancer therapies. Despite major challenges associated with this approach, several compounds that either augment the activity of wild-type p53 or restore all, or some, of the wild-type functions to p53 mutants are currently being explored. In wild-type TP53 cancer cells, p53 function is often abrogated by overexpression of the negative regulator MDM2, and agents that disrupt p53-MDM2 binding can trigger a robust p53 response, albeit potentially with induction of p53 activity in non-malignant cells. In TP53-mutant cancer cells, compounds that promote the refolding of missense mutant p53 or the translational readthrough of nonsense mutant TP53 might elicit potent cell death. Some of these compounds have been, or are being, tested in clinical trials involving patients with various types of cancer. Nonetheless, no p53-targeting drug has so far been approved for clinical use. Advances in our understanding of p53 biology provide some clues as to the underlying reasons for the variable clinical activity of p53-restoring therapies seen thus far. In this Review, we discuss the intricate interactions between p53 and its cellular and microenvironmental contexts and factors that can influence p53's activity. We also propose several strategies for improving the clinical efficacy of these agents through the complex perspective of p53 functionality.

A cytosolic mutp53(E285K) variant confers chemoresistance of malignant melanoma

Malignant melanoma (MM) is known to be intrinsically chemoresistant, even though only ~20% of MM carry mutations of the tumor suppressor p53. Despite improvement of systemic therapy the mortality rate of patients suffering from metastatic MM is still ~70%, highlighting the need for alternative treatment options or for the re-establishment of conventional therapeutic approaches, including chemotherapy. Screening the p53 mutation status in a cohort of 19 patient-derived melanoma samples, we identified one rarely described missense mutation of p53 leading to E285K amino acid exchange (mutp53(E285K)). Employing structural and computational analysis we revealed a major role of E285 residue in maintaining stable conformation of wild-type p53 (wtp53). E285K mutation was predicted to cause interruption of a salt-bridge network affecting the conformation of the C-terminal helix of the DNA-binding domain (DBD) thereby preventing DNA interaction. In this context, a cluster of frequently mutated amino acid residues in cancer was identified to putatively lead to similar structural effects as E285K substitution (E285 cluster). Functional analysis, including knockdown of endogenous p53 and reconstitution with diverse p53 missense mutants confirmed mutp53(E285K) to have lost transcriptional activity, to be localized in the cytosol of cancer cells, by both means conferring chemoresistance. Re-sensitization to cisplatin-induced cell death was achieved using clinically approved compounds aiming to restore p53 wild-type function (PRIMA1-Met), or inhibition of AKT-driven MAPK survival pathways (afuresertib), in both cases being partially due to ferroptosis induction. Consequently, active ferroptosis induction using the GPX4 inhibitor RSL3 proved superior in tumorselectively fighting MM cells. Due to high prevalence of the E285-cluster mutations in MM as well as in a variety of other tumor types, we conclude this cluster to serve an important function in tumor development and therapy and suggest new implications for ferroptosis induction in therapeutic applications fighting MM in particular and cancer in general.

Adoptive Transfer of T Cells Genetically Engineered to Express T-Cell Receptors Targeting Neoantigens Arising from p53 and Ras Hotspot Mutations in Hematologic Malignancies

The tumor suppressor gene p53 and the oncogenes NRAS and KRAS (shortened to Ras) are frequently mutated in many difficult-to-treat hematologic malignancies including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and relapsed multiple myeloma. Driver mutations in p53 and Ras are associated with treatment-refractory malignancies. There are no Food and Drug Administration (FDA)-approved therapies specifically targeting hematologic malignancies with p53 or Ras mutations. Some missense mutations in p53 and Ras lead to immunogenic peptides (neoepitopes) that are presented by human leukocyte antigens (HLA). Members of our group have identified T-cell receptors (TCRs) targeting some of these neoepitopes and used T cells expressing these TCRs as treatments for epithelial tumors (Levin et al. Clin Cancer Res, 2021 and Kim et al. Cancer Immunol Res, 2022). Adoptively transferred T cells expressing some of these TCRs have caused objective responses of advanced epithelial cancers in patients. Here, we report preclinical experiments with T cells genetically engineered to target neoantigens arising from p53 and Ras mutations in hematologic malignancies. p53 mutations occur in approximately 5-10% of de novo AML, 25-50% of treatment-related or complex-karyotype MDS/AML, 5-10% of multiple myeloma, and 25% of plasma cell leukemia. Mutations in Ras occur in 15-20% of AML and 20% of multiple myeloma. Our TCR panel targets neoepitopes arising from 2 of the 5 most common p53 mutation sites and 2 of the 3 most common Ras mutation sites . The human leukocyte antigen (HLA) restriction of these TCRs is shown along with the HLA frequencies ( Table 1). In all experiments, human donor peripheral blood mononuclear cells were activated in culture with an anti-CD3 antibody. Two days later, T cells were transduced with a gamma-retroviral vector encoding a neoepitope-specific TCR. Presence of mutations in target cell lines was confirmed by DNA sequencing. Cell lines which did not naturally express the appropriate HLA were transduced with a gamma-retroviral construct encoding the HLA. We assessed the general specificity of TCRs for each targeted neoepitope. For each neoepitope-specific TCR, T cells expressing the TCR were cultured overnight with target cell lines expressing the neoepitope plus the HLA targeted by that TCR. We cultured T cells from the same cultures with 12 to 16 negative-control target cell lines that lacked the appropriate mutation and/or HLA. Negative control cell lines were from a variety of human tissues. Enzyme-linked immunosorbent assays (ELISA) performed on the culture supernatants showed that IFN-gamma release was much higher when T cells expressing a neoepitope-specific TCR were cultured with target cells having both the targeted mutation and the appropriate HLA (Table 1). We conducted another set of experiments to evaluate the neoepitope-specificity of the TCRs. For each neoepitope, COS7 cells with the appropriate HLA were transfected with plasmids encoding either wild-type or mutant versions of p53 or Ras differing from wild-type by 1 amino acid. The COS7 cells were cultured with T cells expressing the appropriate TCR. IFN-gamma was released in a neoepitope-specific manner. Higher levels of T-cell degranulation and interleukin-2 release occurred when T cells expressing neoepitope-specific TCRs were cultured with target cells having the targeted mutation and HLA versus target cells lacking the appropriate mutation and/or HLA. We evaluated efficacy of the T53-R175H-A2 TCR in vivo. Immunocompromised NOD-scid common gamma-chain-deficient mice received intravenous injections of the KMS26-luciferase multiple myeloma cell line, which naturally expresses HLA-A*02:01 and the p53 R175H mutation. One week later when all mice had detectable tumor burdens, mice received either 10 million R175H-targeted T cells, T cells expressing an irrelevant TCR, or untransduced T cells. All groups received 3 doses of interleukin-2. Mice treated with R175H-targeted T cells cleared the tumor while mice in all other groups had progressive tumor growth (Figure 1). In vivo studies of additional TCRs are in progress. In conclusion, we have demonstrated activity of neoantigen-targeted T cells against hematologic malignancies in preclinical models. We plan to evaluate adoptively transferred autologous T cells targeting p53 and Ras mutations in an upcoming clinical trial.

Mutant p53 sustains serine-glycine synthesis and essential amino acids intake promoting breast cancer growth

Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations.

Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants

Mutations in the TP53 tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specific TP53 missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position—R273C vs. R273H—has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors.

Effect of Vitamin D Supplements on Relapse or Death in a p53-Immunoreactive Subgroup With Digestive Tract Cancer

Recent meta-analyses of randomized clinical trials found that daily vitamin D3 supplementation had beneficial effects on cancer mortality, although the results are still controversial. To examine whether vitamin D supplementation reduces the risk of relapse or death in a supgroup of patients with digestive tract cancer who were p53 immunoreactive. This was a post hoc subgroup analysis of the AMATERASU randomized, double-blind, placebo-controlled clinical trial. This trial included patients at a single university hospital in Japan with digestive tract cancers between January 2010 and February 2018 followed up for a median (IQR) of 3.5 (2.5-5.3) years to compare the effects of vitamin D supplementation with placebo and was reported in 2019. Patients from among 417 participants in the AMATERASU trial whose residual serum samples were available were included. Data were analyzed from October 20 to November 24, 2022. Vitamin D3 (2000 IU/d) supplementation or placebo. The primary outcome was 5-year relapse or death. The subgroup of patients who were p53 immunoreactive was defined by positivity for anti-p53 antibodies in serum and nuclear accumulation of p53 oncosuppressor protein in more than 99% of cancer cells, which is considered a biomarker for p53 missense mutations. Anti-p53 antibody levels were measured using chemiluminescent enzyme immune assay. Immunohistochemical staining data of p53 protein in cancer tissue in pathologic specimens were obtained from a previous study and divided into 4 grades. Among 392 patients with digestive tract cancer (mean [SD] age, 66 [10.7] years; 260 males [66.3%]), there were 37 patients with esophageal cancer (9.4%), 170 patients with gastric cancer (43.4%), 2 patients with small bowel cancer (0.5%), and 183 patients with colorectal cancer (46.7%). Serum anti-p53 antibody was detectable in 142 patients (36.2%), and p53-immunohistochemistry grade showed a positive association with serum anti-p53 antibody levels (coefficient = 0.19; P < .001). In the p53-immunoreactive subgroup (80 patients), relapse or death occurred in 9 of 54 patients (16.7%) in the vitamin D group and 14 of 26 patients (53.8%) in the placebo group; 5-year relapse-free survival (RFS) was significantly higher in the vitamin D group (13 patients [80.9%]) than the placebo group (1 patient [30.6%]; hazard ratio [HR], 0.27; 95% CI, 0.11-0.61; P = .002). This was significantly different from 272 patients in the non-p53 immunoreactive subgroup, in which vitamin D had no effect on 5-year RFS (vitamin D: 35 of 158 patients [22.2%] vs placebo: 24 of 114 patients [21.1%]; HR, 1.09; 95% CI, 0.65-1.84) (P for interaction = .005). This study found that vitamin D supplementation reduced the risk of relapse or death in the subgroup of patients with digestive tract cancer who were p53 immunoreactive. Identifier: UMIN000001977.

Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis

BackgroundTP53, encoding the tumor suppressor p53, is frequently mutated in various cancers, producing mutant p53 proteins (mutp53) which can exhibit neomorphic, gain-of-function properties. The latter transform p53 into an oncoprotein that promotes metastatic tumor progression via downstream effectors such as ENTPD5, an endoplasmic reticulum UDPase involved in the calnexin/calreticulin cycle of N-glycoprotein biosynthesis. Elucidating the mechanisms underlying the pro-metastatic functions of the mutp53-ENTPD5 axis is crucial for developing targeted therapies for aggressive metastatic cancer.MethodsWe analyzed pancreatic, lung, and breast adenocarcinoma cells with p53 missense mutations to study the impact of mutp53 and ENTPD5 on the N-glycoproteins integrin-α5 (ITGA5) and integrin-β1 (ITGB1), which heterodimerize to form the key fibronectin receptor. We assessed the role of the mutp53-ENTPD5 axis in integrin-dependent tumor-stroma interactions and tumor cell motility using adhesion, migration, and invasion assays, identifying and validating therapeutic intervention targets. We employed an orthotopic xenograft model of pancreatic ductal adenocarcinoma to examine in vivo targeting of mutp53-ENTPD5-mediated ITGA5 regulation for cancer therapy.ResultsMutp53 depletion diminished ITGA5 and ITGB1 expression and impaired tumor cell adhesion, migration, and invasion, rescued by ENTPD5. The mutp53-ENTPD5 axis maintained ITGA5 expression and function via the calnexin/calreticulin cycle. Targeting this axis using ITGA5-blocking antibodies, α-glucosidase inhibitors, or pharmacological degradation of mutp53 by HSP90 inhibitors, such as Ganetespib, effectively inhibited ITGA5-mediated cancer cell motility in vitro. In the orthotopic xenograft model, Ganetespib reduced ITGA5 expression and metastasis in an ENTPD5-dependent manner.ConclusionsThe mutp53-ENTPD5 axis fosters ITGA5 and ITGB1 expression and tumor cell motility through the calnexin/calreticulin cycle, contributing to cancer metastasis. ITGA5-blocking antibodies or α-glucosidase inhibitors target this axis and represent potential therapeutic options worth exploring in preclinical models. The pharmacologic degradation of mutp53 by HSP90 inhibitors effectively blocks ENTPD5-ITGA5-mediated cancer cell motility and metastasis in vivo, warranting further clinical evaluation in p53-mutant cancers. This research underscores the significance of understanding the complex interplay between mutp53, ENTPD5, and the calnexin/calreticulin cycle in integrin-mediated metastatic tumor progression, offering valuable insights for the development of potential therapeutic strategies.

Clinicopathological and gene mutation characteristics of uterine carcinosarcoma

To explore the clinicopathological characteristics, immunophenotype, diagnosis and differential diagnosis of uterine carcinosarcoma (UCS), and to explore the gene mutation characteristics and tumor mutation burden (TMB) of UCS. The clinical imaging, pathomorphological data and immunohistochemical expression of 4 cases of UCS, which were archived in the Department of Pathology of the Second Affiliated Hospital of Soochow University from January 2021 to May 2022 were retrospectively analyzed. All exon groups of 4 cases of UCS were sequenced. All the 4 patients were female, aged 47-81 years. The maximum diameter of the tumor was 4.0-13.0 cm, and the boundary was unclear. Microscopically, the tumor was composed of malignant epithelium and sarcoma. Immunohistochemistry showed that the epithelial components of 4 patients expressed broad-spectrum cytokeratin (AE1/E3), the sarcoma components expressed Vimentin, PAX8, ER, PR were expressed to varying degrees, and Ki-67 positive index was high (60%-90%). There were 3 p53 missense mutations, 1 nonsense mutation, 4 MLH1, PMS2, MSH2, MSH6 were positive and PD-L1 was negative. The sequencing results of the whole exon group of 4 UCS patients showed that TP53, BCL9L, BRD4, CLTCLI, PSMD1I, PLEC genes showed a high mutation ratio, which was 3/4, 2/4, 2/4, 2/4, 2/4, 2/4, respectively. TMB analysis showed that the TMB of 4 cases of UCS was<5 mut/Mb. UCS is a rare and highly malignant endometrial tumor. The sequencing results of the whole exon group suggested that TP53, BCL9L, BRD4 and other genes had high mutation rates, suggesting that the occurrence and development of UCS may be closely related to Wnt signaling pathway. Molecular typing indicated that 3 cases of UCS were of high copy number type/p53 mutation type, and 1 case had POLD1 mutation. Microsatellite stability, low PD-L1 expression and TMB results suggested that UCS patients have no obvious advantage in immunotherapy.

DNA-delivered monoclonal antibodies targeting the p53 R175H mutant epitope inhibit tumor development in mice

The tumor suppressor p53 is the most common mutated gene in cancer, with the R175H as the most frequent p53 missense mutant. However, there are currently no approved targeted therapies or immunotherapies against mutant p53. Here, we characterized and investigated a monoclonal antibody (mAb) that recognizes the mutant p53-R175H for its affinity, specificity, and activity against tumor cells in vitro. We then delivered DNA plasmids expressing the anti-R175H mAb or a bispecific antibody (BsAb) into mice to evaluate their therapeutic effects. Our results showed that the anti-R175H mAb specifically bound to the p53-R175H antigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells, with no cross-reactivity with wild-type p53. In cultured cells, the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity. We made a recombinant MC38 mouse cell line (MC38-p53-R175H) that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles. In vivo, administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice. The administration of the anti-R175H BsAb plasmid showed no therapeutic effects, yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody. These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens.

Acetylation halts missense mutant p53 aggregation and rescues tumor suppression in non-small cell lung cancers

TP53 mutations are ubiquitous with tumorigenesis in non-small cell lung cancers (NSCLC). By analyzing the TCGA database, we reported that TP53 missense mutations are correlated with chromosomal instability and tumor mutation burden in NSCLC. The inability of wild-type nor mutant p53 expression can't predict survival in lung cancer cohorts, however, an examination of primary NSCLC tissues found that acetylated p53 did yield an association with improved survival outcomes. Molecularly, we demonstrated that acetylation drove the ubiquitination and degradation of mutant p53 but enhanced stability of wild-type p53. Moreover, acetylation of a missense p53 mutation prevented the gain of oncogenic function observed in typical TP53 mutant-expressing cells and enhanced tumor suppressor functions. Consequently, acetylation inducer targeting of missense mutant p53 may be a viable therapeutic goal for NSCLC treatment and may improve the accuracy of current efforts to utilize p53 mutations in a prognostic manner.

Abstract 1289: The role of mutant p53-mediated mechanisms in modulating the tumor microenvironment and promoting lung metastasis

Abstract INTRODUCTION/METHODS: The multistep process of the tumor invasion-metastasis cascade is a major barrier to effective therapy. Mutations of the tumor suppressor p53 are frequently detected in esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq, ChIP-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer, L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP, in which we have identified macrophage colony stimulating factor 1 (CSF1) to be a direct gene target of p53-R172H. We have targeted CSF1/CSF1R signaling to assess its pro-tumorigenic roles. In addition to studying tumor intrinsic mechanisms, we have established multiplex immunofluorescence and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Overall, this study aims to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote lung metastasis. RESULTS/DISCUSSION: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors and this is dependent upon p53 mutation status, which is reinforced by TCGA data and patient-derived tissue microarrays (TMAs). The TMA analysis also suggested that CSF1 expression is higher in tumors that are poorly differentiated. Furthermore, through the proximity ligation assay (PLA), we have identified that bromodomain and extra-terminal (BET) protein BRD4, which binds to acetylated histones to regulate transcription interacts with p53-R172H at higher levels compared to wild-type p53 to induce Csf1 expression. We show that the BRD4/CSF1/CSF1R signaling axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in lungs in a mutant p53 background. In accordance, targeting CSF1/CSF1R signaling reduces phosphorylation of STAT3 in the metastatic tumors, which suggests a mechanism for increased invasiveness and metastasis. In parallel, upon inhibiting this axis, we have identified decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages, as well as CD31+ endothelial cells, and increased number of CD3+CD8+ cytotoxic T-cells at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of squamous cell carcinoma (SCC) datasets reveals that specific p53 mutations are associated with differential survival rates and CSF1 expression. CONCLUSION: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1-CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other SCCs. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Katherine M. Cunningham, Qiaosi Tang, Kensuke Sugiura, Karen Dunbar, Kensuke Suzuki, Lois Resnick-Silverman, Alison M. Taylor, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. The role of mutant p53-mediated mechanisms in modulating the tumor microenvironment and promoting lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1289.

Abstract 550: p53 rescue of tumor suppressor function with ADGN-531 causes tumor regression both as single agent and in combination with PARPi and KRASG12Cinhibitors

Abstract The p53 tumor suppressor mutations resulting in loss of function control tumor proliferation in 50% of human cancers. TP53 missense and nonsense mutations are present in about 50% and 7-12% of the p53 driven cancer, respectively. To date there is no effective treatment for rescuing p53 function in cancers harboring p53 nonsense mutations. We have developed a new potent strategy combining mRNA with a tumor selective nanocarrier, to rescue of p53 tumor suppressor functions as potential therapeutic approach in cancers. ADGN-531 contains proprietary p53-mRNA complexed with proprietary short amphipathic peptides that form stable neutral nanoparticles. ADGN-531 was evaluated on 20 different cancer cell lines harboring different types of p53 mutations. In-vivo efficacy of IV-administered ADGN-531 nanoparticles (weekly, 0.5 &amp; 1.0mg/kg) was evaluated on colorectal SW403 (p53 nonsense/KRASG12V), osteosarcoma SaOs2 (p53 null) and lung H358 (p53 null/KRASG12C) mouse xenografts. Sensitivity to Veliparib (PARPi) following ADGN-531 treatment was evaluated on PARPi resistant SUM-149PT and OVCAR-8 and PARPi sensitive MDA-MB436 cells. ADGN-531/AMG-510 synergy was evaluated on H358 and M-PACA cells and H358 mouse xenograft. ADGN-531 markedly delay the growth of a large panel of cancer cells harboring p53-missense or nonsense mutations, by rescuing p53 transcriptional activity and subsequent expression of its target proteins. ADGN-531 induces cell cycle arrest in G1 due to p21 upregulation and apoptosis following BAX and PUMA activation. ADGN-531 mediated p53 function rescue is directly correlated to the type of p53 mutation in the cancer cells. ADGN-531 induces 80% cell proliferation inhibition in p53 null/nonsense cells and 20 to 50% in p53 missense cells. IV-administration of ADGN-531 resulted in dose responsive tumor growth inhibition. ADGN-531 (1.0mg/kg) induces 40-60% tumor regression SaOs2, SW403 and H358 xenografted mouse model. We demonstrated a synergistic combination between ADGN-531/AMG-510 a KRASG12C inhibitor in human lung carcinoma. ADGN-531 (0.5mg/kg, weekly)/AMG-510 (10mg/kg, daily) induces about 80% tumor regression, higher than with either single agents. We demonstrated that ADGN-531 mediated p53 rescue markedly improves (200 fold) and restores the sensitivity of ovarian and breast cancer cells to Veliparib. ADGN-531 treatments are well tolerated, no sign of clinical toxicity or inflammatory response was detected after single or repeated administrations. ADGN-531 is effective in rescuing P53 functions both in vitro and in vivo. Our study provides a proof-of-concept that restoration of tumor suppressor function by ADGN-531 could be used as a single agent therapy or in combination together with other therapies for potent combinatorial cancer treatment. Citation Format: Gilles Divita, Audrey Grunenberger, Elodie Czuba, Khadidja Boularache, Melanie Guidetti, Veronique Josserand, Neil Desai. p53 rescue of tumor suppressor function with ADGN-531 causes tumor regression both as single agent and in combination with PARPi and KRASG12Cinhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 550.

Abstract 3950: Sensitizing oncogenic mutant p53-expressing non-small cell lung cancer to proteasome inhibitors

Abstract Background: Lung cancer is the leading cause of cancer deaths worldwide, with non-small cell lung cancer (NSCLC) accounting for ~80% of all lung cancers. Up to 69% of NSCLC are mutated for the tumor suppressor gene TP53, and of these, 84% are missense mutations, where wild-type (WT) TP53 tumor suppressor function is lost and in most cases, emergent cancer-promoting oncogenic activities are acquired. Due to the poor draggability of mutated p53 protein, the focus of therapeutics targeting NSCLC tumors with oncogenic missense mutant p53 has been on vulnerabilities that are not present in normal cells expressing WT p53. One such vulnerability involves oncogenic mutant p53-dependent attenuation of the oxidative stress response. The ability of cancer cells to survive oxidative stress depends on the regulated synthesis and activity of cellular antioxidants, including glutathione (GSH). Proteasome inhibitors (PIs) are known to induce cellular stress at multiple levels, including proteotoxic, autophagic, and oxidative stress, but the clinical utility of PIs has been limited by lack of efficacy in patients with solid tumors, including unselected lung cancer patients. Methods and Results: We now show bortezomib (BTZ) and other PIs are preferentially cytotoxic in oncogenic mutant p53-expressing lung cancer cells in culture. Furthermore, BTZ-treated oncogenic mutant p53-expressing cells display elevated levels of reactive oxygen species and BTZ’s cytotoxic effects were rescued by antioxidants such as N-acetyl cysteine, indicating that oxidative stress is the critical driver of BTZ cytotoxicity. Upon BTZ treatment of oncogenic mutant p53-expressing NSCLC cells, but not in WT p53 expressing NSCLC cells, we also observed the transcriptional induction of pro-apoptotic BH3-only BCL-2 family protein NOXA, consistent with an apoptotic mechanism of BTZ-induced cell death. Furthermore, in combination with BTZ, the use of BH3-mimetics such as venetoclax (ABT-199) or navitoclax (ABT-263) to inhibit the function of pro-survival proteins BCL-2/BCL-XL synergistically enhanced BTZ cytotoxicity, but only in oncogenic mutant p53-expressing NSCLC cells. In vivo mouse xenograft studies demonstrated that combination treatments of BTZ + venetoclax or BTZ + carboplatin (a standard of care chemotherapy for NSCLC) significantly attenuated oncogenic mutant p53-expressing tumor growth. Conclusions: Our data suggest that the preferential cytotoxicity of BTZ, and potentially other PIs, in oncogenic mutant p53-expressing NSCLC cells depends on the induction of toxic oxidative stress leading to NOXA-dependent apoptosis. Thus, combining PIs with drugs that limit antioxidant responses and/or sensitize cells to NOXA-induced apoptosis (e.g., BH3-mimetics) should synergistically and selectively kill NSCLC cells expressing oncogenic mutant p53 and open novel therapeutic avenues for the treatment of NSCLC. Citation Format: Kranthi Kumar Chougoni, Victoria L. Neely, Boxiao Ding, Eziafa Oduah, Khanh Nguyen, Bin Hu, Jennifer Koblinski, Bradford Windle, Hisashi Harada, Steven Grossman. Sensitizing oncogenic mutant p53-expressing non-small cell lung cancer to proteasome inhibitors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3950.

Abstract B014: Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma

Abstract Introduction: Mutations of the tumor suppressor p53 are detected up to 80% of esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates and poor prognosis. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer harboring Trp53R172H/-, in which we have identified Colony stimulating factor 1 (Csf1) to be upregulated. Our goal is to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote tumor invasion and lung metastasis in ESCC. Methods: We have utilized novel L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and isolated tumor cells to model metastatic ESCC. We conducted ChIP-Seq analysis for p53 on tumor cells derived from lung metastases in our mouse models. Furthermore, we have genetically and pharmacologically targeted CSF1/CSF1R signaling axis to assess its role in ESCC tumor invasion and lung metastasis. In addition to studying tumor intrinsic mechanisms, we have established quantitative multiplex immunofluorescence (qmIF) and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Results And Discussion: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors, and this is dependent upon p53 mutation status, which is reinforced by the TCGA data and patient-derived tissue microarrays (TMAs). The overlay of the RNA-Seq with the ChIP-Seq analysis indicates that Csf1 is a direct gene target of p53-R172H with enriched binding motifs. Furthermore, based on the proximity ligation assay (PLA) with murine ESCC tumor cells, bromodomain and extra-terminal motif (BET) protein BRD4 interacts with p53-R172H to induce Csf1 expression. We show that the BRD4-CSF1 axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in a mutant p53 background. In accordance, upon inhibiting this signaling pathway in the tail-vein injection lung metastasis models, we have identified reduced expression of CD31+ cells and decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of the esophageal cancer datasets reveals that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. Based upon these results, we are generating human and murine esophageal cells with distinct DNA contact and conformational p53 mutations via base editing. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1/CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other squamous cell cancers. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Qiaosi Tang, Katherine M. Cunningham, Kensuke Sugiura, Karen Dunbar, Kausik Regunath, Kensuke Suzuki, Andres J. Klein-Szanto, Lois Resnick-Silverman, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B014.