P53 Mutations In Tumors Research Articles

Mutant p53 drives an immune cold tumor immune microenvironment in oral squamous cell carcinoma

The critical role of the tumor immune microenvironment (TIME) in determining response to immune checkpoint inhibitor (ICI) therapy underscores the importance of understanding cancer cell–intrinsic mechanisms driving immune-excluded (“cold”) TIMEs. One such cold tumor is oral cavity squamous cell carcinoma (OSCC), a tobacco-associated cancer with mutations in the TP53 gene which responds poorly to ICI therapy. Because altered TP53 function promotes tumor progression and plays a potential role in TIME modulation, here we developed a syngeneic OSCC models with defined Trp53 (p53) mutations and characterized their TIMEs and degree of ICI responsiveness. We observed that a carcinogen-induced p53 mutation promoted a cold TIME enriched with immunosuppressive M2 macrophages highly resistant to ICI therapy. p53-mutated cold tumors failed to respond to combination ICI treatment; however, the combination of a programmed cell death protein 1 (PD-1) inhibitor and stimulator of interferon genes (STING) agonist restored responsiveness. These syngeneic OSCC models can be used to gain insights into tumor cell–intrinsic drivers of immune resistance and to develop effective immunotherapeutic approaches for OSCC and other ICI-resistant solid tumors.

Carrier-free nanoprodrug for p53-mutated tumor therapy via concurrent delivery of zinc-manganese dual ions and ROS.

Human cancers typically express a high level of tumor-promoting mutant p53 protein (Mutp53) with a minimal level of tumor-suppressing wild-type p53 protein (WTp53). In this regard, inducing Mutp53 degradation while activating WTp53 is a viable strategy for precise anti-tumor therapy. Herein, a new carrier-free nanoprodrug (i.e., Mn-ZnO2 nanoparticles) was developed for concurrent delivery of dual Zn-Mn ions and reactive oxygen species (ROS) within tumor to regulate the p53 protein for high anti-tumor efficacy. In response to the mild tumor acidic environment, the released Zn2+ and H2O2 from Mn-ZnO2 NPs induced ubiquitination-mediated proteasomal degradation of Mutp53, while the liberative Mn2+ and increased ROS level activated the ATM-p53-Bax pathway to elevate WTp53 level. Both in vitro and in vivo results demonstrated that pH-responsive decomposition of Mn-ZnO2 NPs could effectively elevate the intracellular dual Zn-Mn ions and ROS level and subsequently generate the cytotoxic hydroxyl radical (•OH) through the Fenton-like reaction. With the integration of multiple functions (i.e., carrier-free ion and ROS delivery, tumor accumulation, p53 protein modulation, toxic •OH generation, and pH-activated MRI contrast) in a single nanosystem, Mn-ZnO2 NPs demonstrate its superiority as a promising nanotherapeutics for p53-mutated tumor therapy.

Abstract 723: Dual inhibition of MDM2 and p38 MAPK signaling is a potential therapeutic strategy in esophageal squamous cell carcinoma

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal malignancies. Despite recent advances in treatment, the overall 5-year survival rate remains low, primarily due to late diagnosis, acquired resistance to therapy, and distant metastases. Hence, development of novel therapeutic strategies is required to improve patient outcomes. The MDM2 gene is amplified in some patients with ESCC, frequently decreasing the tumor suppressive function of p53. In addition, 40-60% of ESCC tumors exhibit p53 mutations. Missense mutations within the p53 gene cause its overexpression and subsequent activation of p38 MAPK signaling. In this study, we aimed to investigate the effect of combining a p38 MAPK inhibitor and an MDM2 antagonist as a potential therapeutic strategy in ESCC. Methods: To determine the synergistic anti-tumor activity of the MDM2 antagonist siremadlin (HDM-201) and the p38 MAPK inhibitor Ralimetinib dimesylate (LY2228820), we performed cell viability, colony formation, apoptosis, and cell cycle analysis in ESCC cell lines. Next, we evaluated the anti-tumor activity of HDM-201 and LY2228820 in a xenograft mouse model. Finally, we performed siRNA mediated silencing of p53 to assess its functional role in the MDM2 antagonist-induced activation of p38 MAPK signaling and its ability to modulate the DNA damage response (DDR) by measuring ROS status and phosphorylation of H2AX. Results: HDM-201 exerted cytotoxic effects in p53-mutated ESCC cells in a dose-dependent manner and activated the p38 MAPK signaling pathway. These results prompted us to combine the p38 MAPK inhibitor LY2228820 with HDM-201 and evaluate this novel treatment strategy in a p53-mutant pre-clinical model of ESCC. We observed that HDM-201 and LY2228820 acted synergistically to decrease cell viability (p<0.01) and colony formation (p<0.01), as well as induction of apoptosis (p<0.01) and G2/M phase cell cycle arrest (p<0.01). The xenograft animal model confirmed our in vitro findings wherein the combined treatment exerted more potent anti-tumor activity (p<0.05). We also observed that silencing of p53 promoted resistance towards the MDM2 antagonist and did not induce apoptosis, highlighting that a functional p53 is required for inducing apoptosis after combined treatment. Furthermore, we observed that the combined treatment promoted ROS induced oxidative stress (p<0.01) and promoted DNA double strand breaks (DSBs) as evidenced by increased phosphorylation of H2AX (p<0.01), highlighting its potential as an effective therapeutic strategy. Conclusion: We provide novel pre-clinical evidence that inhibition of p38 MAPK signaling can augment the anti-tumor activity of an MDM2 antagonist in p53-mutant human tumor models of ESCC. Citation Format: Souvick Roy, Rachana Garg, David Wang, Ajay Goel. Dual inhibition of MDM2 and p38 MAPK signaling is a potential therapeutic strategy in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 723.

MEDB-33. The landscape of ecDNA in medulloblastoma

Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive cancers, including medulloblastoma (MB), the most common malignant pediatric brain tumor. To assess the clinical importance of ecDNA in MB, we applied computational methods to detect ecDNA in the genomes of a cohort of 468 MB patients and 31 MB model systems. Among patients, ecDNA was detected in 18% of tumors and carried a threefold greater risk of mortality. Affected genomic loci harbor up to hundredfold amplification of oncogenes including MYC, MYCN, TERT, and other novel putative oncogenes. Between sequential patient biopsies at initial diagnosis and subsequent relapse, we observed structural variation at ecDNA loci and generation of new ecDNA sequences. Among model systems, ecDNA was found in 19 of 31 genomes (61%). Although ecDNA was far more prevalent among MB models than patients, the ecDNA genomic sequences were conserved between most patient-derived xenograft (PDX) models and the human tumors from which they were made. To elucidate the functional regulatory landscapes of ecDNAs in MB, we generated transcriptional (RNA-seq), accessible chromatin (ATAC-seq), and chromatin interaction (Hi-C) profiles of 6 MB tumor samples. In each case, we identified regulatory interactions that cross fusion breakpoints on the ecDNA, representing potential “enhancer rewiring” events which may contribute to transcriptional activation of co-amplified oncogenes. To test this hypothesis, we are currently conducting in vitro CRISPRi screens targeting regulatory regions on the ecDNA of a MB cell line to determine whether these enhancers promote proliferation. Using single-cell sequencing, we have also begun exploring intratumoral heterogeneity of ecDNA in a p53-mutant SHH MB patient tumor and its corresponding PDX model. In summary, our study analyzes the frequency, diversity, and functional relevance of ecDNA across MB subgroups and provides strong justification for continued mechanistic studies of ecDNA in MB with the potential to uncover new therapeutic approaches.

Drugging the undruggable-targeting P53: A comparative analysis of APR-246 and coti-2 in human tumor primary culture explants.

e15092 Background: The TP53 tumor suppressor is the most commonly mutated gene in human cancer. P53 protein comprised of 393 amino acids functions as a tetramer with the loss of function mutations concentrated in “hotspots” that often affect DNA binding domains. APR-246 and COTI-2 are novel small molecules that are under clinical investigation for the treatment of P53 mutated cancers. APR, a quinuclidine binds to P53 cysteine residues altering the functional conformation while COTI is a thio-semicarbazone, found active in P53 mutant tumors using a novel computational platform. As the mechanisms of action may differ we undertook a comparative analysis of these compounds in 3D human tumor explants derived from surgical specimens. Methods: Ex Vivo Analyses of Programmed Cell Death (EVA/PCD) (D’Amora, P et al Gyn. Oncol. 2021) was applied to 236 human tumor primary culture explants isolated from surgical specimens. After disaggregation, micro-spheroids, isolated by precise density centrifugation were exposed to APR, COTI and other agents for 72-96 hours. Drug-induced cell death was measured by delayed-loss-of-membrane integrity and ATP content (luciferase). Dose response curves were interpolated to LC50 values. Synergy analysis used the method of Chou & Talalay. Correlation coefficients used Pearson moment. Results: APR and COTI reveal the highest activity in hematologic neoplasms. Solid tumor activity patterns diverged with COTI active in gynecologic malignancies and NSCLC and moderately active in breast and colorectal while APR is active in colorectal but less active in breast and NSCLC. Pearson moments for COTI vs APR revealed an R value = 0.146 (NS). COTI activity correlated with Nitrogen Mustard and Alpelisib (PIK3CA) while APR activity correlated with Doxorubicin and Everolimus. COTI combinations focused upon ovarian cancers identified synergy in 25% and antagonism in 18%. Conclusions: P53’s function as the “guardian of the genome” is consistent with our findings that many cytotoxic and targeted agents that induce cell death reveal correlations with APR and COTI. While APR affects the structural conformation of P53, COTI may function downstream via oxidative, metabolic or other stress responses. The correlation coefficient for APR vs COTI at 0.146 (NS) suggests distinct mechanisms of action. This is supported by different disease-specific-profiles and correlations with other drugs. COTI synergy in ovarian cancers, the target of current clinical trials at 25% is below expectations. With differing disease specificities, selecting between these and related agents to optimize clinical outcomes may require a more granular examination of cellular mechanisms of response and resistance. Primary culture analyses offer insights into drug mechanisms of action, combinatorial potential and have the capacity to improve patient selection.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Ultrasound-triggered reactive oxygen species effector nanoamplifier for enhanced combination therapy of mutant p53 tumors

Reactive oxygen species (ROS) damage is a crucial method with which to inhibit tumor cell proliferation; however, tumor cells can reduce ROS damage by modulating multiple repair mechanisms, thus, reducing the efficacy of ROS damage in tumor therapy. In this study, we built an ultrasound-triggered ROS damage nanoamplifier using a synergistic strategy consisting of ROS damage and decreased tumor self-protection capability to enhance the treatment efficacy of mutant p53 tumors. A ROS damage nanoamplifier (PT@PTGA) was fabricated using amphiphilic polyglutamic acid (PTGA) to load with a sonosensitizer (protoporphyrin IX, PpIX) and an MTH1 inhibitor (TH287). Under ultrasonic excitation, PpIX catalyzes oxygen to produce singlet oxygen and release TH287 to inhibit MTH1 activity, thereby causing the accumulation of 8-oxo-dGTP, which enhances DNA damage and further induces cell apoptosis. In addition, TH287 allies with ROS to eliminate the mutated p53 protein in tumor cells, thus reducing the self-protective capacity of tumor cells. As a result, the "internal and external" aspects were combined to enhance sensitization for mutant p53 tumor therapy. The construction of a ROS nanoamplifier not only provides an effective strategy for the treatment of mutant p53 tumors but also supplies an integrated platform for tumor diagnosis and therapy.

Enterobacteria impair host p53 tumor suppressor activity through mRNA destabilization

Increasing evidence highlights the role of bacteria in the physiopathology of cancer. However, the underlying molecular mechanisms remains poorly understood. Several cancer-associated bacteria have been shown to produce toxins which interfere with the host defense against tumorigenesis. Here, we show that lipopolysaccharides from Klebsiella pneumoniae and other Enterobacteria strongly inhibit the host tumor suppressor p53 pathway through a novel mechanism of p53 regulation. We found that lipopolysaccharides destabilize TP53 mRNA through a TLR4-NF-κB-mediated inhibition of the RNA-binding factor Wig-1. Importantly, we show that K. pneumoniae disables two major tumor barriers, oncogene-induced DNA damage signaling and senescence, by impairing p53 transcriptional activity upon DNA damage and oncogenic stress. Furthermore, we found an inverse correlation between the levels of TLR4 and p53 mutation in colorectal tumors. Hence, our data suggest that the repression of p53 by Enterobacteria via TLR4 alleviates the selection pressure for p53 oncogenic mutations and shapes the genomic evolution of cancer.

Abstract P5-09-03: Mieap, a p53-downstream gene, is associated with suppression of breast cancer cell proliferation and better survival

Abstract (Introduction) We previously reported that the mitochondria-eating protein (Mieap), a p53-downstream gene, is involved in mitochondrial quality control (MQC) by repairing or eliminating unhealthy mitochondria. In the breast mass, endogenous cytoplasmic Mieap was detected in 32% of invasive ductal carcinomas (IDCs), 55.6% of ductal carcinomas in situ, and 88.9% of fibroadenomas by immunohistochemistry (IHC). We also demonstrated that the p53/Mieap-regulated MQC pathway was inactivated in 26.1% of IDCs, wherein its impairment resulted in a significantly shorter disease-free survival (DFS) of patients with primary breast cancer. Therefore, in the present study, we aimed to characterize Mieap as a tumor suppressor and determine its association with prognosis using large human breast cancer cohorts. (Materials and methods) We identified 2980 patients from two worldwide large-scale primary breast cancer cohorts: The Cancer Genome Atlas (TCGA; n=1076) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n=1904). Mieap expression and survival data were extracted using the cBioPortal and Broad Institute Firehose websites. Regarding the ER+/HER2- (luminal), HER2+, and ER-/HER2- (triple negative; TN) breast cancer subtypes, 1336, 236, and 267 patients from the METABRIC cohort were found to have the respective subtypes, whereas from the TCGA cohort, 584, 182, and 157 patients had the respective subtypes. We then divided patients into the high and low groups on the basis of median gene expression levels in each cohort and analyzed the association between Mieap expression and clinical outcome. The Kaplan-Meier method was used to compare the survival curves between Mieap-high and Mieap-low groups, and Fisher’s exact test was performed to compare the clinicopathological characteristics for significance. Furthermore, expression of Mieap-associated genes was evaluated by Gene Set Enrichment Analysis (GSEA). (Results) Mieap expression was significantly downregulated in tumors than in normal tissues in TCGA. It was significantly downregulated in p53-mutant tumors in the whole cohort, and in all luminal, HER2+, and TN subtypes of both cohorts, indicating that Mieap expression was dependent on p53 status. Furthermore, BNIP3 and NIX, co-factors of Mieap, were downregulated in tumors than in normal tissues in TCGA. These expressions were positively correlated with Mieap expression. GSEA demonstrated that cell cycle- and proliferation-associated gene sets (Myc Targets, E2F targets, G2M checkpoint, Mitotic spindle etc.) were significantly enriched in Mieap-low tumors than in Mieap-high tumors. Mieap expression was significantly correlated with KI67, a cell proliferation marker. Mitochondria-associated gene sets were enriched in Mieap-high tumors. Mieap was found to be downregulated in advanced cancer stage and/or histological grade consistently in both cohorts. Mieap expression was downregulated in metastatic tumors than in primary tumors. In the METABRIC cohort, we found that the DFS and overall survival of patients with Mieap-high tumors were significantly longer than those of patients with Mieap-low tumors among those with the whole and luminal subtypes. Similarly, in the TCGA cohort, the DFS of patients with Mieap-high tumors was longer among those with the luminal subtype. (Conclusion) In conclusion, we found that the mechanistic role of Mieap was clinically relevant in the two independent cohorts. Specifically, Mieap was found to be p53-dependent, and its downregulation was associated with advanced breast tumors. Moreover, Mieap-high tumors demonstrated favorable breast cancer prognosis, particularly in the luminal subtype, because of Mieap’s control over the cell cycle. Thus, Mieap plays an important role as a tumor suppressor in breast cancer. Citation Format: Manabu Futamura, Yoshihisa Tokumaru, Kazuaki Takabe, Hirofumi Arakawa, Kazuhiro Yoshida. Mieap, a p53-downstream gene, is associated with suppression of breast cancer cell proliferation and better survival [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-03.

Abstract P5-04-04: Identification of AR driven tumors within TNBC using a novel gene signature

Abstract Background The androgen receptor (AR) is an emerging biomarker and favourable therapeutic target in breast cancer (BC), especially in Triple negative BC (TNBC) which lacks a targeted treatment and shows high molecular heterogeneity. Proportion of AR positive tumours are known to vary (10 to 43%) within TNBC and recent multi-institutional study on AR positive TNBC, showed AR protein status alone is not a reliable prognostic marker for therapeutic response. A deeper examination of AR regulated pathways is necessary to derive AR regulated gene signatures which could identify TNBC tumours truly driven by AR and amenable to anti androgen therapies. In this study, we attempted to develop AR regulated gene signature using gene expression data derived from cell lines under the influence of androgens using systematic bioinformatic approach and tested the ability of the signature to identify AR regulated tumours within TNBC. Methods Microarray data of AR positive BC cell lines (GSE61368 & GSE28305) was used to derive AR regulated genes by a bioinformatic pipeline, using integrated method by incorporating protein interaction networks, topological parameters, and semantic similarity to arrive at final set of 16 genes. Correlation of these genes with AR protein status by reverse phase protein array (RPPA) was evaluated in 447 tumors of TCGA dataset. A 3rd quartile cut-off in the AR expression (by RPPA) was used to classify them as AR positive and negative and a logistic regression model was constructed using positive AR as determinant to generate AR probability score with minimum set of genes. 3rd quartile cut-off in the probability score was used to classify tumors as AR high and low. Distribution of AR probability score was examined in 144 and 187 TNBC tumors in TCGA and METABRIC, respectively. Comparisons between AR high and low tumors were done for clinical parameters, mutational spectrum, expression of ER regulated genes (FOXA1, GATA3 and TFF1) and EMT gene signatures (Byers et al., 2013, Tan et al., 2014). Luminal androgen receptor (LAR) subtype identification by TNBCtype (Lehmann et. al) was done in both datasets to examine the overlap with our method of identification of AR driven tumors within TNBC. Results Logistic regression model identified 5 (SOCS2, ID1,PIP,GADD45 and ZNF689) of the 16 AR regulated genes as predictors for AR probability score which ranged from 0.009 to 0.68 in 144 TNBC tumors within TCGA data. 41/144 (29%) TNBC tumors were categorized as having AR high at cut off of 0.13. The AR probability score ranged from 0.04 to 0.64 in 187 TNBC tumors within METABRIC dataset and 47/187 (25%) were classified as AR driven tumors at cut off of 0.22. Though differences in the clinical variables like age , tumor size and lymph node(LN) status were not different between the AR high and low groups in the TCGA dataset, a significant increase in age, LN positive tumors was observed in the AR high groups in METABRIC. A significant higher PIK3CA mutations was observed in the AR high tumors both in the TCGA and METABRIC (p=0.012 and p<0.0001 respectively) with a proportionate lower p53 mutations in the AR high tumors in both data sets (p=0.03 and p=0.35 respectively). Further, AR high tumors showed significant higher expression of ER regulated genes (TCGA-p<0.05 & METABRIC, p<0.05), were more epithelial by EMT score (TCGA-p<0.05 & METABRIC, p<0.05). Analysis by TNBCtype showed that 30% of AR high tumors in TCGA and 40% of AR high tumors in METABRIC were identified as belonging to LAR subgroup (Lehmann et al). Conclusion Identification of AR driven tumors within TNBC has both prognostic and predictive utility. Methods using limited number of AR regulated genes could be easily applied to larger BC cohorts to identify TNBC tumours driven by AR signalling and may respond well to anti-androgen therapies. Citation Format: Savitha Rajarajan, Snijesh V.P, Maalavika Pillai, Mohit Kumar Jolly, Jyothi S Prabhu. Identification of AR driven tumors within TNBC using a novel gene signature [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-04-04.

P53 and EGFR gene mutations in malignant tumors of the lung

Relevance: Increased incidence of lung cancer globally and in Kazakhstan, lack of screening in hereditary cases, high mortality, and low survival of patients necessitate the study of the molecular genetic causes of the disease. At present, gene mutation studies for lung cancer diagnostics are expanding. However, many gene mutations revealed remain undercovered in the
 scientific literature, and there is not enough data on their prognostic and diagnostic value.
 The purpose of the study was to discover the specifics of the р53 gene mutations and reveal the EGFR exon 19 deletions and exon 21 L858R mutations in malignant tumors of the lung of various histogenesis.
 Methods: The mutations were studied in tumors (200 samples) and adjacent tissue (200 samples) of patients with lung cancer (squamous cell carcinoma (SCC) and adenocarcinoma (ADC) of the lung) by polymerase chain reaction (PCR), electrophoresis, and EcoR1- and Pst1-restriction of samples after p53 gene fragments and cDNA amplification and mRNA revertase treatment. Another 263 lung cancer samples were evaluated by real-time PCR for EGFR exon 19 deletions and EGFR exon 21
 L858R mutations.
 Results: The p53 gene was not expressed in 50% of SCC and adenocarcinoma of the lung samples. Restriction revealed p53 mRNA mutations in 100% of SCC and 75% of ADC samples. p53 exon-intron 5-6 was mutated in 50% of ADC and 70% of SCC samples, exon-intron 7-9 – in 60% of SCC cases. EGFR exons 19 and 21 mutations found in 65 of 263 lung tumor samples were associated with increased sensitivity to EGFR tyrosine kinase inhibitors.
 Conclusion: The p53 gene mutations revealed in most samples of SCC and ADC of the lung could be used to diagnose lung cancer and predict its severity. The identified EGFR mutations allow predicting the effectiveness of targeted therapy.

A Quantitative Systems Approach to Define Novel Effects of Tumour p53 Mutations on Binding Oncoprotein MDM2.

Understanding transient protein interactions biochemically at the proteome scale remains a long-standing challenge. Current tools developed to study protein interactions in high-throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self-assembling protein microarrays produced by freshly translating full-length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label-free platform that measures many protein interactions in real-time allowing the determination of the KDs and rate constants. The developed novel NAPPA-SPRi technique showed excellent ability to study protein-protein interactions of clinical mutants of p53 with its regulator MDM2. Furthermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin-3, currently in clinical practice, which usually disrupts the p53-MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg-273-Cys, Arg-273-His, Arg-248-Glu, Arg-280-Lys), on the structural domain (His-179-Tyr, Cys-176-Phe), on hydrophobic moieties in the DNA binding domain (Arg-280-Thr, Pro-151-Ser, Cys-176-Phe) and hot spot mutants (Gly-245-Cys, Arg-273-Leu, Arg-248-Glu, Arg-248-Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities.

A Personalized Neoantigen Vaccine in Combination with Platinum-Based Chemotherapy Induces a T-Cell Response Coinciding with a Complete Response in Endometrial Carcinoma.

Simple SummaryWe investigated the feasibility and immunogenicity of an autologous Dendritic Cell (DC) vaccine pulsed with peptide neoantigens in combination with a standard of care regimen. The vaccine program took place in a serous endometrial mismatch repair (MMR) deficiency setting. We demonstrate for the first time the feasibility of producing a peptide DC vaccine in endometrial carcinoma. The safety and immunogenicity of this personalized vaccine was demonstrated by the detection of polyfunctional and durable T-cell responses.Endometrial cancer (EC) is a common gynecological malignancy and the fourth most common malignancy in European and North American women. Amongst EC, the advanced serous, p53-mutated, and pMMR subtypes have the highest risk of relapse despite optimal standard of care therapy. At present, there is no standard of care maintenance treatment to prevent relapse among these high-risk patients. Vaccines are a form of immunotherapy that can potentially increase the immunogenicity of pMMR, serous, and p53-mutated tumors to render them responsive to check point inhibitor-based immunotherapy. We demonstrate, for the first time, the feasibility of generating a personalized dendritic cell vaccine pulsed with peptide neoantigens in a patient with pMMR, p53-mutated, and serous endometrial adenocarcinoma (SEC). The personalized vaccine was administered in combination with systemic chemotherapy to treat an inoperable metastatic recurrence. This treatment association demonstrated the safety and immunogenicity of the personalized dendritic cell vaccine. Interestingly, a complete oncological response was obtained with respect to both radiological assessment and the tumor marker CA-125.

Abstract 1015: mTOR inhibitor induces non-apoptotic, autophagy dependent cell death (ADCD) in TP53 mutant HNSCC

Abstract TP53 is the most frequently mutated gene in Head and Neck Squamous Cell Carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis. A phase II multi-institutional clinical trial, determined that, adjuvant therapy with an mTOR inhibitor significantly increased 2-year progression free survival (PFS) in patients with p53 mutant tumors. Previous studies have shown that mutant p53 causes sustained activation of PI3K/AKT/mTOR pathway in cancer. We investigated the mechanism by which the mTOR inhibitor CCI-779 inhibits cell growth of TP53 mutant HNSCC in vitro. HPV-negative HNSCC cell lines harboring TP53 mutations at different positions were treated with CCI-779.CCI-779 significantly reduced cell viability and colony forming ability of all the cell lines tested. CCI-779 did not induce apoptosis or cell cycle arrest in these cells. However, the reduction in cell viability was associated with increase in autophagy as demonstrated by increased levels of LC3-II. The autophagy inhibitors, 3-methyl adenine (3-MA) and ULK-101 restored the cell viability by inhibiting CCI-779 induced autophagy. Therefore, we postulate that CCI-779 induces nonapototic autophagy dependent cell death (ADCD) in HPV-negative TP53 mutant HNSCC cell lines. CCI-779 induced autophagy is accompanied by increased level of ULK1 and Beclin1 as well as increased phosphorylation of ULK1 at Serine 555. However, ULK101 (ULK1 inhibitor) and 3-MA (PI3K inhibitor) when combined with CCI-779 reduced the level of both total and phosphorylated levels of ULK1, which resulted in inhibition of autophagy and improved cell viability. However, combination of CCI-779 and bafilomycin A1 treatment showed increased level of LC3-II, ULK1 and phospho-ULK1 (S555) with concomitant decrease in cell viability. Our data suggests that rapalog induces ADCD by facilitating autophagy initiation through the stabilization and activation of ULK1. Moreover our data demonstrated that CCI-779 mediated activation of ULK1 is associated with down regulation of mTORC1 but not mTORC2 signaling. This is a novel mechanism of how rapalogues induce ADCD through the stabilization/activation of ULK1 in HPV-negative TP53 mutant HNSCC cells. Together these data highlight the importance of how mTORi can be used to accelerate tumor cell death in HPV-negative TP53 mutant HNSCC. Citation Format: Md Maksudul Alam, Janmaris Marin Fermin, Patrick T. Spiller, Channing Dorr, Xiaohua Rong, Tara-Moore Medlin, Alok R. Khandelwal, Cherie-Ann Nathan. mTOR inhibitor induces non-apoptotic, autophagy dependent cell death (ADCD) in TP53 mutant HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1015.

Abstract 2494: Identification of novel oncogenic targets of mutant p53 in esophageal squamous cell carcinoma

Abstract Background: Mutations in the TP53 gene are observed at a high frequency in Esophageal Squamous Cell Carcinoma (ESCC) with a majority of alterations being of the missense type affecting residues located in the region encoding the DNA binding domain. These mutations are associated with poor patient survival. However, characterization of the transcriptional networks regulated by p53 mutant proteins is limited to only a few frequent ‘hotspot' mutants, while comparatively rarer mutants have not been characterized. Objectives: Identification and characterization of differentially expressed genes associated with p53 mutation status in ESCC tumor samples. Elucidation of the mechanism of transcriptional regulation of mutant p53 target genes. Methods: A defined set of ESCC tumor samples with known p53 mutation status was selected for this study. The ability of each 'rare' p53 mutation to modulate tumor-related phenotypes was evaluated by performing phenotypic assays such as proliferation, colony formation and migration, in head and neck cancer cell-lines. Gene expression microarray analysis was performed on 36 ESCC tumors to determine putative target genes of 'rare' p53 mutant forms. Following dimension reduction, differentially expressed (putative mutant p53 target) genes were identified using Significance Analysis of Microarrays (SAM) and validated using reverse transcription quantitative PCR (RT-qPCR). Transcription induction of the putative target genes by ectopically expressed wild-type and mutant p53, in p53-null H1299 cell-line, were also evaluated by RT-qPCR. Chromatin immuno-precipitation (ChIP) assay was used to determine localization of mutant p53 proteins to chromosomal loci harboring the putative target genes. Similarly, ability to activate target promoters by mutant p53 was validated through promoter-luciferase assay. Results: Genome-wide analysis revealed 27 differentially expressed genes, of which 9 exhibited up-regulated transcript levels in p53 mutant tumors. Three putative mutant p53 target genes - C1QBP, ARF6 and TRIM23 - were selected for further analysis due to previous reports of their association with cancer. TP53 transcript levels positively correlated with transcript levels of the identified potential mutant targets. The ectopically-expressed p53 mutants - P190T and P278L, induced the increased expression of the target genes, were localized to and activated the promoters of the target genes. Further characterization of the target genes is currently underway. Conclusions: Three novel potential oncogenic targets of mutant p53 proteins were identified. The study further strengthened the heterogeneity in the functioning of different p53 mutants. Citation Format: Sara A. George, Viswakalyan Kotapalli, Raju S. Adduri, Raju Kumar, Sanjana Sarkar, Ramaswamy Pandilla, Murali D. Bashyam. Identification of novel oncogenic targets of mutant p53 in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2494.

Abstract 1284: P53-independent restoration of p53 pathway in tumors with mutated p53 through ATF4 transcriptional modulation

Abstract A long-term goal in the cancer-field has been to develop strategies for treating p53-mutated tumors. A novel small-molecule, PG3-Oc, restores p53 pathway-signaling in tumor cells with mutant-p53, independently of p53/p73. PG3-Oc partially upregulates the p53-transcriptome (13.7% of public p53 target-gene dataset; 15.2% of in-house dataset) and p53-proteome (18%, HT29; 16%, HCT116-p53-/-). Bioinformatic analysis indicates critical p53-effectors of growth-arrest (p21), apoptosis (PUMA, DR5, Noxa), autophagy (DRAM1), and metastasis-suppression (NDRG1) are induced by PG3-Oc. ERK1/2- and CDK9-kinases are required to upregulate ATF4 by PG3-Oc which restores p53 transcriptomic-targets in cells without functional-p53. PG3-Oc represses MYC (ATF4-independent), and upregulates PUMA (ATF4-dependent) in mediating cell death. With largely nonoverlapping transcriptomes, induced-ATF4 restores p53 transcriptomic targets in drug-treated cells including functionally important mediators such as PUMA and DR5. Our results demonstrate novel p53-independent drug-induced molecular reprogramming involving ERK1/2, CDK9, and ATF4 to restore upregulation of p53 effector genes required for cell death and tumor suppression. Citation Format: Xiaobing Tian, Nagib Ahsan, Wafik S. El-Deiry. P53-independent restoration of p53 pathway in tumors with mutated p53 through ATF4 transcriptional modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1284.

Natural Merosesquiterpenes Activate the DNA Damage Response via DNA Strand Break Formation and Trigger Apoptotic Cell Death in p53-Wild-type and Mutant Colorectal Cancer.

Simple SummaryBowel cancer is a serious disease, which affects many people worldwide. Unfortunately, the disease is often diagnosed in an advanced stage, which impairs the chance of survival. Furthermore, resistance to therapy occurs frequently. Thus, novel therapeutic approaches are required to improve cancer therapy. Here, we studied whether merosesquiterpenes might be useful for cancer treatment. These compounds occur in marine sponges and were isolated by our group. We were able to identify three compounds with potent cytotoxic activity in different cell lines established from human large bowel cancer. Our experiments provided evidence that the compounds cause DNA damage and trigger cell death, so-called mitochondrial apoptosis, which was attested in cancer cells with expression of wild-type and mutated p53 tumor suppressor. Finally, we show that merosesquiterpenes also kill intestinal tumor organoids, an ex vivo model of large bowel cancer.Colorectal cancer (CRC) is a frequently occurring malignant disease with still low survival rates, highlighting the need for novel therapeutics. Merosesquiterpenes are secondary metabolites from marine sponges, which might be useful as antitumor agents. To address this issue, we made use of a compound library comprising 11 isolated merosesquiterpenes. The most cytotoxic compounds were smenospongine > ilimaquinone ≈ dactylospontriol, as shown in different human CRC cell lines. Alkaline Comet assays and γH2AX immunofluorescence microscopy demonstrated DNA strand break formation in CRC cells. Western blot analysis revealed an activation of the DNA damage response with CHK1 phosphorylation, stabilization of p53 and p21, which occurred both in CRC cells with p53 knockout and in p53-mutated CRC cells. This resulted in cell cycle arrest followed by a strong increase in the subG1 population, indicative of apoptosis, and typical morphological alterations. In consistency, cell death measurements showed apoptosis following exposure to merosesquiterpenes. Gene expression studies and analysis of caspase cleavage revealed mitochondrial apoptosis via BAX, BIM, and caspase-9 as the main cell death pathway. Interestingly, the compounds were equally effective in p53-wild-type and p53-mutant CRC cells. Finally, the cytotoxic activity of the merosesquiterpenes was corroborated in intestinal tumor organoids, emphasizing their potential for CRC chemotherapy.

Differential chromatin accessibility landscape of gain-of-function mutant p53 tumours

BackgroundMutations in TP53 not only affect its tumour suppressor activity but also exerts oncogenic gain-of-function activity. While the genome-wide mutant p53 binding sites have been identified in cancer cell lines, the chromatin accessibility landscape driven by mutant p53 in primary tumours is unknown. Here, we leveraged the chromatin accessibility data of primary tumours from The Cancer Genome Atlas (TCGA) to identify differentially accessible regions in mutant p53 tumours compared to wild-type p53 tumours, especially in breast and colon cancers.ResultsWe identified 1587 lost and 984 gained accessible chromatin regions in breast, and 1143 lost and 640 gained regions in colon cancers. However, only less than half of those regions in both cancer types contain sequence motifs for wild-type or mutant p53 binding. Whereas, the remaining showed enrichment for master transcriptional regulators, such as FOX-Family TFs and NF-kB in lost and SMAD and KLF TFs in gained regions of breast. In colon, ATF3 and FOS/JUN TFs were enriched in lost, and CDX family TFs and HNF4A in gained regions. By integrating the gene expression data, we identified known and novel target genes regulated by the mutant p53.ConclusionThis study reveals the direct and indirect mechanisms by which gain-of-function mutant p53 targets the chromatin and subsequent gene expression patterns in a tumour-type specific manner. This furthers our understanding of the impact of mutant p53 in cancer development.

RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence

SummaryRecent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer.

Cost-effectiveness analysis of p53 immunohistochemical testing in stage I and II high-risk endometrial cancer.

e18838 Background: PORTEC-3, a phase III clinical trial comparing adjuvant radiation alone (RT) to combined chemoradiation therapy (CTRT) in patients with high-risk endometrial cancer (EC), demonstrated no benefit with CTRT compared to RT for patients with stage I and II EC. However, a subsequent tumor molecular analysis revealed that individuals with stage I and II high-risk EC with p53 mutations had statistically significantly improved progression-free survival (PFS) and clinically significantly improved overall survival (OS) with combined CTRT. As p53 immunohistochemical (IHC) testing and CTRT adds to cost and toxicity, we sought to evaluate the cost-effectiveness of p53 IHC tumor testing in patients with stage I and II high-risk EC. Methods: A Markov decision model was developed to compare two testing strategies in patients with stage I and II high-risk EC: p53 IHC testing vs. no IHC testing. IHC staining for p53 is an accurate and validated surrogate for TP53 mutational status. As a result, p53 IHC testing alone was used in the decision analysis. Data from PORTEC-3 and the subsequent molecular analysis were used for the base case model parameters, including treatment regimen, PFS, OS, and incidence of p53-mutated tumors. Cost and utility data were derived from the Federal Supply Schedule, the Centers for Medicare and Medicaid Services (CMS) Fee Schedules, the Tufts CEA registry, peer-reviewed literature, and expert input. Data analysis was performed using TreeAge Pro Software, 2021. Effectiveness outcomes included quality-adjusted life-years (QALYs) and incremental cost-effectiveness ratios (ICERs). Sensitivity analyses were performed in order to identify the influence of individual variables on the overall model. Results: When compared to no testing, p53 IHC testing resulted in a cost savings of $471 per patient while providing an additional 0.310 QALYs. Therefore, p53 IHC testing was less expensive and more effective than no IHC testing and was a dominant strategy in this model. In one-way sensitivity analyses, CTRT and RT costs, discount rate, and percentage of p53 mutations detected had the most impact on the model; however, p53 IHC testing remained a cost-saving strategy over all parameter ranges examined. Conclusions: For patients with high-risk stage I and II EC, this analysis suggests that p53 IHC testing is cost-effective compared to no testing in determining the costs and benefits of adjuvant chemotherapy. Although additional studies are warranted, this data provides further support for the role of molecular-based treatment decisions for high-risk stage I and II endometrial cancer.