P53 R273H Research Articles

Hepatitis B Virus X Protein Induces Reactive Oxygen Species Generation via Activation of p53 in Human Hepatoma Cells.

Hepatitis B virus (HBV), particularly through the HBx protein, induces oxidative stress during liver infections. This study reveals that HBx increases reactive oxygen species (ROS) via two distinct mechanisms. The first mechanism is p53-independent, likely involving mitochondrial dysfunction, as demonstrated by elevated ROS levels in p53-deficient Hep3B cells and p53-knocked-down HepG2 cells after HBx expression or HBV infection. The increase in ROS persisted even when p53 transcriptional activity was inhibited by pifithrin-α (PFT-α), a p53 inhibitor. The second mechanism is p53-dependent, wherein HBx activates p53, which then amplifies ROS production through a feedback loop involving ROS and p53. The ability of HBx to elevate ROS levels was higher in HepG2 than in Hep3B cells. Knocking down p53 in HepG2 cells lowered ROS levels, while ectopic p53 expression in Hep3B cells raised ROS. HBx-activated p53 downregulated catalase and upregulated manganese-dependent superoxide dismutase, contributing to ROS amplification. The transcriptional activity of p53 was crucial for these effects, as cells with a p53 R175H mutation or those treated with PFT-α generated less ROS. Additionally, HBx variants with Ser-101 increased p53 and ROS levels, whereas variants with Pro-101 did not. These dual mechanisms of HBx-induced ROS generation are likely significant in the pathogenesis of HBV and may contribute to liver diseases, including hepatocellular carcinoma.

Abstract C071: Transcription factor switching drives progression of the classical subtype of pancreatic cancer

Abstract Despite recent advances in the treatment of pancreatic adenocarcinoma (PDAC), the median survival remains <12 months. Patients typically present with late-stage disease and are often unable to tolerate drug combination regimens. Large-scale genomic approaches categorise PDAC into two distinct molecular subtypes, based on transcriptomic signatures, and these are termed the classical and squamous (or basal) subtypes. Although it is well-described that PDAC harbours gene mutations (i.e. KRAS G12D, p53 R172H) that initiate the disease, little is known about the influence of lineage-defining Transcription Factors (TF) on PDAC subtypes and their contribution to tumor progression. We took an unbiased approach to discover driving TFs in PDAC, we performed ChIP-seq of the active enhancer mark H3K27Ac from surplus tissue following resection of primary PDAC tumors (n=6) and normal adjacent tissue (n=4). Analysis of the H3K27Ac sites showed that the most enriched motifs within all experimentally mapped enhancer elements were HNF motifs (HNF4G & HNF4A) and Forkhead (i.e., FOXA1), confirming that these two classes of TFs constitute the lineage-defining factors in these clinical samples. Investigation of publicly available clinical datasets revealed HNF4G to be amplified in 9% of PDAC patients. Using Rapid Immunoprecipitation and Mass Spectrometry of Endogenous protein complexes (RIME), our method for unbiased discovery of endogenous protein complexes, we investigated the fundamental differences in the FOXA1 interactome between models of classical and squamous subtypes. HNF4A and HNF4G were only seen as FOXA1-interactors in models of the classical subtype. HNF4G Knockout (KO) clones injected orthotopically into the pancreas of mice, showed a significant increase (p-value<0.0006) in median survival and slower tumor growth compared to Control (Cas-9) (p-value <0.0001). HNF4G-KO dependent genes were specifically expressed in primary tumors (with decreased EMT signalling) versus metastatic samples (PanCRux dataset), implying that the HNF4G target genes were mostly limited to primary tumor contexts. We found Protein Arginine Methylate Transferase (PRMT1) to be a significant and reproducible interactor of FOXA1 and HNF4G. We observed a significant global reduction in PRMT1 chromatin binding in both the HNF4G-KO and FOXA1 depleted cells compared to the control. This data suggests a dependency on both HNF4G and FOXA1 for recruitment of PRMT1 to chromatin. We find HNF4G to be the key driver of classical subtype primary disease, in part by recruiting the methyltransferase PRMT1 to regulatory regions revealing a therapeutically exploitable opportunity. A molecular switch occurs in advanced disease, whereby HNF4G expression decreases, unmasking FOXA1’s transcriptional activity. De-repressed FOXA1 drives late-stage disease, by orchestrating metastasis-specific enhancer-promoter loops to direct metastatic gene regulation. We suggest the existence of stage-dependent TF activity, triggered by molecular compartmentalisation that mediates multistage progression of PDAC. Citation Format: Shalini Vasudev Rao, Lisa V Young, Danya Cheeseman, Stephanie V Mack, Dominique-Laurent Couturier, Sean V Flynn, Rebecca Brias, Amy Smith, Jill Temple, Duncan Jodrell, Alasdair Russell V Russell, Igor Chernukhin, Jason Carroll. Transcription factor switching drives progression of the classical subtype of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C071.

Abstract C064: Mapping cellular plasticity through the lens of FRA1 during Kras G12D mediated pancreatic acinar to ductal metaplasia

Abstract Introduction: Acinar cells have been proposed as a cell-of-origin for pancreatic ductal adenocarcinoma (PDAC) after undergoing acinar to ductal metaplasia (ADM). ADM can be triggered by pancreatitis causing acinar cells to de-differentiate to a ductal-like state. We identify FRA1 (gene name Fosl1) as the most active transcription factor during Kras G12D acute pancreatitis-mediated injury. Herein, we explore the role of the FRA1 in the onset of ADM and neoplastic transformation. Methods: We generated tamoxifen (TAM)-inducible Ptf1aCre ERT ;Kras G12D ; Rosa26 YFP/YFP (TAM-KCY Fosl1 WT ) mice and bred in the Fosl1 fl/fl allele to create TAM- KCY Fosl1 KO mice and subjected them to caerulin (CCK analog) induced acute pancreatitis. First, we performed a time course of hematoxylin and eosin (H&E) images from mice either with Fosl1 WT or Fosl1 KO after induction of acute pancreatitis to understand the functional role of FRA1 during acinar de-differentiation. Next, after snap-freezing pancreata at the Day 5 post-caerulein timepoint and extracting nuclei, we sorted YFP+ nuclei for snATAC sequencing and bulk-RNA sequencing. We developed a pseudotime model and gene-regulatory network (GRN) to understand the dynamic landscape as cells transitioned from normal acinar to late ADM. Finally, to determine the functional role of in tumorigenesis, we bred an inducible mutant p53 R172H gain- of-function allele into TAM-KCY Fosl1 WT/KO mice to create TAM-KPCY Fosl1 WT/KO mice. These mice were subjected to the same tamoxifen and acute pancreatitis protocol as TAM-KCY Fosl1 WT/KO mice and monitored for tumor growth and overall survival. Results: A time course of H&E images demonstrated that FRA1 depletion slowed progression to maximum ADM in the first 5 days after caerulein treatment. We additionally observed a dramatic reduction in the amount of PanIN lesions formed between TAM-KCY Fosl1 WT and TAM-KCY Fosl KO mice, which occurred on day 7 in this caerulein model. Pseudotime analysis proposed a time point when FRA1 becomes active to prime ADM cells for recovery from inflammation and simultaneously undergo PanIN transformation. Furthermore, GRN analysis validated the differentially active transcription factors identified by pseudotime analysis of snATAC-seq. To elaborate on further timepoints, we observed a delay in the progression of TAM-KCY Fosl KO PanIN lesions at later Day 21, 8-week and 12-week timepoints. In the survival analysis, TAM-KPCY Fosl1 WT mice had a median survival time of 25.86 weeks, while TAM-KPCY Fosl1 KO mice survived for almost twice as long with a median survival time of 40.57 weeks. Histologically, the percentage of Ki-67- positive cells in TAM-KPCY Fosl1 WT tumors was significantly higher than in TAM-KPCY Fosl1 KO tumors, suggesting the loss of FRA1 reduce tumor aggressiveness. Conclusions: Our findings reveal that FRA1 is a critical mediator of ADM progression to PanIN and PDAC. Fosl1 genetic depletion can alter the kinetics of ADM formation and lead to a decrease in PanIN and eventual malignant transformation. Citation Format: Alina Li, Noriyuki Nishiwaki, Kensuke Sugiura, Kensuke Suzuki, Constanza Nicole Tapia Contreras, Dorsay Sadeghian, Anirban Maitra, Jason R Pitarresi, Rohit Chandwani, Peter A Sims, Anil K Rustgi. Mapping cellular plasticity through the lens of FRA1 during Kras G12D mediated pancreatic acinar to ductal metaplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C064.

Mutant p53 Exploits Enhancers to Elevate Immunosuppressive Chemokine Expression and Impair Immune Checkpoint Inhibitors in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective treatments. It is characterized by activating KRAS mutations and p53 alterations. However, how these mutations dysregulate cancer-cell-intrinsic gene programs to influence the immune landscape of the tumor microenvironment (TME) remains poorly understood. Here, we show that p53R172H establishes an immunosuppressive TME, diminishes the efficacy of immune checkpoint inhibitors (ICIs), and enhances tumor growth. Our findings reveal that the upregulation of the immunosuppressive chemokine Cxcl1 mediates these pro-tumorigenic functions of p53R172H. Mechanistically, we show that p53R172H associates with the distal enhancers of the Cxcl1 gene, increasing enhancer activity and Cxcl1 expression. p53R172H occupies these enhancers in an NF-κB-pathway-dependent manner, suggesting NF-κB's role in recruiting p53R172H to the Cxcl1 enhancers. Our work uncovers how a common mutation in a tumor-suppressor transcription factor appropriates enhancers, stimulating chemokine expression and establishing an immunosuppressive TME that diminishes ICI efficacy in PDAC.

Proteasome inhibition paradoxically degrades gain-of-function mutant p53 R273H in NSCLC and could have therapeutic implications.

Lung cancer is the leading cause of cancer mortality. Despite therapeutic advances in recent years, new treatment strategies are needed to improve outcomes of lung cancer patients. Mutant p53 is prevalent in lung cancers and drives several hallmarks of cancer through a gain-of-function oncogenic program, and often predicts a poorer prognosis. The oncogenicity of mutant p53 is related to its stability and accumulation in cells by evading degradation by the proteasome. Therefore, destabilization of mutant p53 has been sought as a therapeutic strategy, but so far without clinical success. In this study, we report that proteasome inhibition results in degradation of mutant p53 in non-small cell lung cancer (NSCLC) cell lines bearing the R273H mutant protein and show evidence that this was mediated by hsp70. NSCLC cell lines with the mutant R273H allele demonstrated increased susceptibility and apoptosis to proteasome inhibitors. These data suggest that proteasome inhibitors could have therapeutic implications in some subsets of TP53 mutated NSCLC.

Abstract 10: Non-viral engineered T cell therapy specific for the hotspot mutation p53 R175H that integrates signal 1 (TCR), signal 2 (co-stimulation) and signal 3 (cytokine) and co-opts FasL-dependent apoptosis to achieve a coordinated antitumor CD4/8 T cell response

Abstract Adoptive T cell therapy (ACT) has demonstrated antitumor efficacy in patients with solid cancers but requires further optimization to become a reproducibly effective treatment. T cell receptor (TCR)-engineered T cells recognize peptides derived from intracellular and surface proteins presented in the context of MHC class I. Targeting mutated oncogenic drivers addresses many of the major obstacles of this modality, in that the antigenic epitope is: 1) tumor-specific, 2) essential for tumor survival, and 3) derived from a stably expressed protein. However, the immune-suppressive tumor microenvironment makes further optimization of engineered T cells necessary to bring long-term clinical benefit to patients. For an optimal anti-tumor response, T cells require three signals: TCR, co-stimulation, and cytokine signaling. The tumor suppressor TP53 is the most frequently mutated gene across human cancers, with a highly recurrent arginine to histidine hotspot alteration in codon 175 leading to novel tumor-dependent functions. Here we report the use of a novel CRISPR-Cas nuclease system to knock-in a six-parameter multi-cistronic cassette into the TRAC locus with high efficiency. We employed several strategies to maximize the potency and durability of a TCR-T cell product targeting the p53 R175H oncogenic driver, including: 1) A high-affinity TCR (α and β chains) specific for the p53 R175H mutation presented by HLA-A*02:01 permits the recognition of tumor cells expressing even low levels of the epitope (Signal 1), 2) Inclusion of the CD8αβ co-receptor drives stimulation of CD4+ T cells with the MHC class I restricted TCR, allowing for a physiologic coordinated immune response required for maximal efficacy, 3) A FAS-41BB switch receptor acts as a dominant negative to the FASL-inducing apoptotic signal in the tumor microenvironment and drives stimulation and persistence of the T cell product via 41BB co-stimulatory signaling (Signal 2), 4) A chimeric cytokine receptor (constitutive Interleukin Receptor) promotes expansion and survival while avoiding immunologic exhaustion (Signal 3). Together, these strategies deliver the three signals required for maximal T cell function: antigen-driven activation, co-stimulation, and growth/survival-promoting cytokine signaling. The non-viral TRAC-knocked-in T cells demonstrate robust and specific cytotoxicity against endogenously expressing HLA-A*02:01 and p53 R175H cell lines in vitro and effective anti-tumor activity in vivo while maintaining a favorable preclinical safety profile. These data support the planned clinical development of a novel non-viral TRAC-knocked-in T cell therapy for the treatment of p53 R175H-mutant solid tumors. Citation Format: Santosh Narayan, Ken Gareau, Ankit Gupta, Josh Ferrell, Nicholas Rouillard, Tyler Warner, Jinsheng Liang, Luhua Shen, Tanya Tetrault, Joshua Francis, Xingyue He, Patrick J. Browne, Rebecca Lamothe, Meghan D. Storlie, Gregory J. Cost, Thomas M. Schmitt, Philip D. Greenberg, Smita S. Chandran, Damien Hallet, Michael Gormally, Chistopher A. Klebanoff, Gary Shapiro, Kim Nguyen, Loïc Vincent. Non-viral engineered T cell therapy specific for the hotspot mutation p53 R175H that integrates signal 1 (TCR), signal 2 (co-stimulation) and signal 3 (cytokine) and co-opts FasL-dependent apoptosis to achieve a coordinated antitumor CD4/8 T cell response [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 10.

Abstract B043: Dual epigenetic therapy combined with anti-PD1 rescues HMA-induced suppressive myeloid phenotype and reduces tumor growth in a PDAC model

Abstract INTRODUCTION. We previously showed, using the PDAC mouse model KPC (KrasLSL G12D/+; p53 r172H/+; Pdx1-Cre), that sequential treatment with the DNA hypomethylating drug decitabine (5aza-dC; DAC) followed by anti-PD-1 led to increased tumor necrosis, slower tumor growth, increased tumor-infiltrating CD8+ and CD4+ T-cells (TILs), and significantly increased mean survival time. However, despite these positive outcomes, the KPC tumors eventually escaped from treatment. We identified a unique myeloid population as a specific subtype of M2-polarized putatively immunosuppressive Chil3+ macrophages (TAMs). Here we further characterize the recruitment mechansism of these cells and show that HDAC inhibition can further improve the efficacy of DAC+PD-1 treatment. METHODS. Primary macrophages were isolated from bone marrow (BMDM) or the peritoneum of 6-8 weeks mice and RAW264.7 cells were also used and exposed to conditioned media from primary KPC cell lines after HMA treatment (KPC-SN-DAC). Gene set enrichemnt analysis was done on RNASeq data from myeloid cells and validated by RT-qPCR. We used cytokine arrays to identify secreted cytokines from KPC cells. KPC cells were orthtopically injected in C57BL/6 mice, and tumor growth was followed by untrasound. Treatments were started when tumor reaced a size of 1 mm3. RESULTS. Expression profiling reveled that exposure of the myeloid cells to KPC-SN-DAC resulted in an HMA-specific M2-like gene signature. Notably DAC alone had minimal effect on myeloid cells polarization. To assess which factors might trigger this polarization we employed cytokine arrays and identified a number of candidate cytokines induced or secreted from KPC cells specifically following HMA treatment. In order to revert this unfavorable cytokine profile, we have evaluated the combination of DAC with four different HDAC inhibitors with reported immunomodulatory effects: Romidepsin (ROMI), Domatinostat (DOMA), Pracinostat (PRAC) and Entinostat (ENT). In vitro synergy studies through cell viability assays showed that ROMI, PRAC and DOMA synergized at low doses with DAC. Pofiling of supernatants from KPC cells treated with a combination of DAC+HDACis showed that, while ROMI had limited effect, PRAC and DOMA reverted DAC-induced cytokine secretion. Lastly, we tested in vivo the combination of DAC+ROMI/DOMI/PRAC+PD1 using mice orthotopically injected with KPC cells. The combination DAC+PRAC+PD1 showed reduced tumor volume compared to DAC+PD1. Moreover, PRAC+PD1 significantly improved overall survival. Interestingly, HDAC inhibition rescued the increased Chil3 expression in DAC+PD1 treated tumors. CONCLUSIONS. Our results show that paracrine rather than direct effect of hypomethylating therapy is responsible for suppressive myeloid polarization and characterize a specific effect of hypomethylating therapy on immature myeloid cells. Moreover, we have identified HDAC inhibition as a viable strategy to prevent unfavorable myeloid polarization and improve the therapeutic efficacy of the DAC+PD1 paradigm. Citation Format: Arturo Orlacchio, Stephen Muzyka, Catherine Do, Diane M. Simeone, Tamas Gonda. Dual epigenetic therapy combined with anti-PD1 rescues HMA-induced suppressive myeloid phenotype and reduces tumor growth in a PDAC model [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 B043.

Abstract C080: Decoding the MYC-PDAC nexus: Unveiling the impact of PP2A-C subunit methylation on tumor onset and advancement

Abstract Introduction: Persistent disruptions in MYC regulation and the inactivation of the serine/threonine protein phosphatase 2A (PP2A) are prevalent among individuals diagnosed with pancreatic ductal adenocarcinoma (PDAC). While it is known that PP2A influences MYC activity by facilitating its degradation, the precise mechanism by which this occurs remains unclear. Small molecule activators PP2A (SMAPs) regulate the holoenzyme assembly consisting of a scaffolding-A subunit and a catalytic-C subunit, and over 40 distinct substrate directing regulatory-B subunits. PP2A heterotrimerization is regulated by post-translational modifications of the catalytic C subunit, notably methylation of L309, which impacts B subunit binding affinity to the AC dimer and thus the substrate selectivity. In this study, we aim to investigate whether the deregulation of MYC, attributed to the loss of PP2A-C subunit methylation, contributes to the initiation and progression of PDAC. Methods: We investigated the influence of SMAPs on MYC’s stability using clinical specimens, cellular, and mouse models. Clinical specimens consisted of tissue microarray (TMA) from patients with pancreatitis and PDAC. Cellular models involved ex-vivo acinar cell assays and proximity ligation assays. Mouse models included caerulein-induced pancreatitis, genetic mouse models like Ptf1a-Cre; LSL-KRASG12D (KC) and LSL-Kras G12D;P53 R172H/+;Pdx1-Cre (KPC), along with pharmacological inhibition studies with SMAPs. Results and discussion: Our TMA data revealed a consistent pattern of Methyl-PP2A-C (Methyl-C) loss and an increase in the stable and active phosphoform of MYC (p62MYC) throughout the sequential stages of PDAC progression, starting from normal tissue and advancing to acinar-to-ductal metaplasia, PanINs, and finally PDAC. In ex-vivo ADM assays, treatment with small molecule activators of PP2A (SMAPs), specifically DT061, significantly reduced the transformation of acinar cells to duct-like cells compared to untreated cells from both wild-type (WT) and KC mice. This suggests that PP2A plays a crucial role in the early events that drive the formation of pancreatic lesions. Moreover, SMAPs demonstrated the ability to alleviate caerulein-induced inflammation in normal mice, as well as suppress the development of pancreatic PanIN lesions and collagen deposition in caerulein-treated KC mice. These effects were associated with decreased levels of p62MYC and increased Methyl-C staining in both WT and KC mice treated with caerulein. Additionally, in KPC mice, treatment with DT061 resulted in reduced PDAC tumor growth, accompanied by decreased levels of p62MYC. Conclusion: Our results provide compelling evidence that small molecule activators of PP2A (SMAPs), through their ability to downregulate MYC, hold significant promise as a therapeutic strategy for individuals at high risk of developing PDAC, particularly those with chronic pancreatitis. This approach has the potential to effectively prevent PDAC development and mitigate its onset in this high-risk population. Citation Format: Vidhi Shah, Alexander Smith, Gabriel Cohn, Motoyuki Tsuda, Goutham Narla, Jonathan Brody, Brett Sheppard, Rosalie Sears. Decoding the MYC-PDAC nexus: Unveiling the impact of PP2A-C subunit methylation on tumor onset and advancement [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 C080.

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.

Biological Effects of HDAC Inhibitors Vary with Zinc Binding Group: Differential Effects on Zinc Bioavailability, ROS Production, and R175H p53 Mutant Protein Reactivation.

The coordination of zinc by histone deacetylase inhibitors (HDACi), altering the bioavailability of zinc to histone deacetylases (HDACs), is key to HDAC enzyme inhibition. However, the ability of zinc binding groups (ZBGs) to alter intracellular free Zn+2 levels, which may have far-reaching effects, has not been explored. Using two HDACis with different ZBGs, we documented shifts in intracellular free Zn+2 concentrations that correlate with subsequent ROS production. Next, we assayed refolding and reactivation of the R175H mutant p53 protein in vitro to provide greater biological context as the activity of this mutant depends on cellular zinc concentration. The data presented demonstrates the differential activity of HDACi in promoting R175H response element (RE) binding. After cells are treated with HDACi, there are differences in R175H mutant p53 refolding and reactivation, which may be related to treatments. Collectively, we show that HDACis with distinct ZBGs differentially impact the intracellular free Zn+2 concentration, ROS levels, and activity of R175H; therefore, HDACis may have significant activity independent of their ability to alter acetylation levels. Our results suggest a framework for reevaluating the role of zinc in the variable or off-target effects of HDACi, suggesting that the ZBGs of HDAC inhibitors may provide bioavailable zinc without the toxicity associated with zinc metallochaperones such as ZMC1.

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.

Abstract LB095: Hybrid TCR-CAR design surpasses conventional CARs and patient-derived TCRs in targeting an ultra-low-density neoantigen

Abstract Mutation-associated neoantigens (MANAs) are exquisitely cancer-specific therapeutic targets. However, MANAs are present at ultra-low densities on the cancer cell surface (as few as 1-2 copies per cell), leading to the challenge of eliciting a sufficiently robust therapeutic effect. We combined components of both T cell receptors (TCRs) and chimeric antigen receptors (CARs) to create a new receptor with improved potency against an ultra-low-density MANA. From CARs, we utilized the antibody-based antigen recognition domain (i.e. the single chain variable fragment, scFv) and the integrated co-stimulation that amplifies T cell activation. From TCRs, we utilized the multi-chain signaling platform that facilitates high antigen sensitivity. This new receptor, termed a TCR Embedded ScFv for Long-term Activation (TESLA), showed promising characteristics when tested with the H2-scFv which targets the p53 R175H mutation presented on HLA-A*02:01 (R175H/A2). Using CRISPR-based homology directed repair in primary human T cells, we tested 15 configurations of appending the H2-scFv to subunits of the TCR complex to identify a design that maximized T cell cytotoxicity and interferon gamma release in co-cultures with cancer cells expressing endogenous levels of the R175H/A2 antigen. In this system, we showed that the optimal TCR-embedded configuration of the H2-scFv produced similar levels of cytotoxicity and interferon gamma secretion as patient-derived TCRs targeting the same R175H/A2 MANA, while conventional H2-CARs were unable to produce any T-cell activation. We then used a multiple stimulation co-culture system to identify a co-stimulation domain combination (MyD88 and CD40) that improved serial cytotoxicity and proliferation of H2-TESLAs when incorporated on the intracellular side of the TCRbeta chain. Finally, we compared the H2-TESLA receptor to patient-derived TCRs modified with the same MyD88 and CD40 co-stimulation domains. In vivo, H2-TESLAs cured all mice in a tumor model, while co-stimulation-modified TCRs produced only temporary tumor control. Moreover, in vivo, H2-TESLAs elicited 100-fold greater T cell expansion than co-stimulation-modified TCRs. In conclusion, we demonstrated that by combining aspects of both CARs and TCRs, the TESLA receptor improved T cell reactivity against an ultra-low-density neoantigen compared to conventional CARs and patient-derived TCRs. Citation Format: Brian J. Mog, Sarah R. DiNapoli, Michael S. Hwang, Tushar D. Nichakawade, Jacqueline Douglass, Emily Han-Chung Hsiue, Katharine M. Wright, Alexander H. Pearlman, Maximilian F. Konig, Suman Paul, Nicolas Wyhs, Nikita Marcou, Stephanie Glavaris, Jiaxin Ge, Michelle S. Miller, P. Aitana Azurmendi, Evangeline Watson, Drew M. Pardoll, Sandra B. Gabelli, Chetan Bettegowda, Nickolas Papadopoulos, Kenneth W. Kinzler, Bert Vogelstein, Shibin Zhou. Hybrid TCR-CAR design surpasses conventional CARs and patient-derived TCRs in targeting an ultra-low-density neoantigen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB095.

Abstract 619: TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV associated oral squamous cell carcinoma

Abstract Background: TP53 is the most common mutated gene in head and neck cancer, including oral squamous cell carcinoma (OSCC), a lethal disease with limited therapeutic options. In OSCC, for example, TP53 R175H is a hot-spot, high-risk, functional mutation that inhibits the function of WT p53, however these correlations and functional roles of high-risk p53 mutations had not been experimentally addressed using syngeneic oral tumor models. To understand the functional role of TP53 mutations in modulating the tumor immune microenvironment (TIME) and response to immunotherapy, we used a syngeneic oral tumor model (ROC3 cell) derived from a genetically engineered mouse model expressing the p53 R172H (human, R175H). Methods: Orthotopic tongue injections were performed to (i) characterize tumor growth rate (ii) the TIME landscape by flow cytometry and opal multiplex immunofluorescence (mIF); and (iii) immunotherapy drug studies. We inactivated mutant p53 expression by shRNA and CRISPR Cas9, profiled gene expression changes by HTG EdgeSeq array, and validated these findings by qPCR, Western blot and immunochemistry. Tumor response was achieved by anti-PD1 and STING agonist therapies. Patients’ gene expression analyzed by CIBERSORT and TIME landscape performed by mIF. The spatial distribution of TIME was evaluated by Visiopharm software and UMAP. Results: We demonstrated that disruption of mutant p53R172H in ROC3 cells altered cytokine and chemokine expression levels and consequently affected the maturation and infiltration of different immune subsets, including cytotoxic CD8+ T cells, helper CD4+ T cells, Tregs, G-MDSCs, tumor-associated macrophages and neutrophils in ROC3 tumors. In addition, our spatial TIME analysis revealed that p53R172H mutation promoted the infiltration of Tregs, M2 macrophages, and exhausted CD8+ T cells, and excluded active CD8+ and CD11+ cells in both the tumor cores and on the tumor edges. Interestingly, it seems that p53R172H also regulates the spatial distribution of immunocyte populations, and their distribution between central and peripheral intratumoral locations. Moreover, we found that p53R172H mutation not only disrupted the phosphorylation of STING pathway which supported the recently reported finding, but also decreased the absolute amount of STING protein. Furthermore, we detected that p53-mutated tumor-infiltrating CD8+ and CD4+ T cells expressed programmed cell death protein 1 (PD-1), and that these tumors responded to anti-PD-1 and STING agonist therapy. Furthermore, Human OSCC samples also revealed associations between immune cell populations and the TP53R175H mutation, which paralleled the findings from our ROC3 tumor model. Conclusions: Our findings collectively demonstrate that the mutant TP53 GOF mutation modulates the tumor immune landscape and is associated with reduced overall survival in patients with OSCC. Citation Format: Yewen Shi, Xiaoyong Ren, Shaolong Cao, Tongxin Xie, Xi Chen, Chieko Michikawa, Abdullah A. Osman, Andrew G. Sikora, Jeffrey N. Myers, Roberto Rangel. TP53 gain-of-function mutation modulates the immunosuppressive microenvironment in non-HPV associated oral squamous cell carcinoma [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 619.

Abstract C041: Hypomethylating therapy induces a potential immuno-suppressive myeloid phenotype by altering cancer cell cytokine secretion in PDAC

Abstract Introduction: We have previously shown using the KPC (KrasLSL G12D/+; p53 r172H/+; Pdx1-Cre) mouse model of PDAC that sequential treatment with the DNA hypomethylating agents (HMA) followed by anti-PD-1 led to increased tumor necrosis, slowed tumor growth, increased tumor-infiltrating CD8+ cells, and significantly increased mean survival. However, acquired treatment resistance occurred, with emergence of a specific subtype of M2-polarized putatively immunosuppressive Chi3l3+ macrophages. In this study, we characterize the mechanism of polarization of these cells, define their function, and identify a potential therapeutic strategy to combine with epigentic therapy to prevent unfavourable macrophage polarization and improve the efficacy of epigenetic priming of immunotherapy against PDAC. Methods: Studies were conducted with primary macrophages (PM)e isolated from bone marrow (BMDM) or the peritoneum of 6-8 weeks mice and RAW264.7 cells were also used and exposed to conditioned media from primary KPC cell lines with and without previous treatment with the HMAs decitabine or azacytidine. The effect was compared with polarization by direct HMA, IFN-γ and IL-4. Gene set enrichment analysis was done on RNASeq data from myeloid cells and validated by RT-PCR. We used cytokine arrays and Western blot validation to identify secreted cytokines from KPC cells. Results: We profiled expression of RAW264.7 cells, BMDM and PMs following exposure to IL-4 or IFN-γ and identified a signature associated with each treatment (CD206, Fizz1, Tlr8, IGF1, Mgl2 associated with IL-4 and TNFα, CD86, CD64, CD40, NOS2 associated with IFN-γ). Direct exposure of myeloid cells to hypomethylating agents did not result in a significant polarization. We next used conditioned media of KPC cells to identify a hypomethylation specific effect and found a significant IL-4 like enrichment (Chi3l3, Arg1, Il4i1, Raet1a, Lgals4) and a HMA-specific myeloid signature (CD137, IL1a, Ccl2, Ccl7, Spp1) with treatment. To assess which cytokines might trigger this polarization, we employed cytokine arrays and identified a small number of candidate cytokines that are specific to hypomethylation induced polarization of myeloid cells, including CXCL1/2, CCL2, GM-CSF, FGF-21, IGFBP-6 and ICAM-1. Conclusions: Our results show that paracrine secretion of cytokines from cancer cells treated with HMA drugs, rather than direct effect of hypomethylating therapy on macrophages, is responsible for polarizing macrophages into an immunosuppressive subtype. We further identified several candidate cytokines secreted by cancer cells following hypomethylating therapy that may be relevant for this immunosuppressive polarization of macrophages and might be specific therapeutic targets in combination with hypomethylating therapy. Citation Format: Arturo Orlacchio, Daniel Weissinger, Catherine Do, Benjamin Tycko, Diane M. Simeone, Tamas Gonda. Hypomethylating therapy induces a potential immuno-suppressive myeloid phenotype by altering cancer cell cytokine secretion in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C041.

LncRNA H19 Promotes Lung Adenocarcinoma Progression via Binding to Mutant p53 R175H.

Simple SummaryThis research explored the association and interaction between lncRNA H19 and mutant p53 (R175H) in lung adenocarcinoma development and progression. H19 over-expression may induce the elevated expression of mtp53 and interact with mtp53, which prolongs the p53 half-life and promotes transcriptional activity, leading to the progression of lung adenocarcinoma. The simultaneous inhibition of H19 and mtp53 may provide a novel strategy.Background: Accumulating data suggest that long non-coding RNA (lncRNA) H19 and p53are closely related to the prognosis of lung cancer. This study aims to analyze the association and interaction betweenH19 and mutant p53 R175H in lung adenocarcinoma (LAC). Methods: Mutant-type (Mt) p53 R175H was assessed by using RT-PCR in LAC cells and 100 cases of LAC tissue samples for association with H19 expression. Western blot, RNA-pull down, immunoprecipitation-Western blot and animal experiments were used to evaluate the interaction between H19 and mtp53. Results: Mtp53 R175H and H19 were over-expressed in LAC tissues and cells, while H19 over-expression extended the p53 half-life and enhanced transcriptional activity. Combined with anti-p53, ShH19 can significantly inhibit tumor growth in vivo. Conclusions: H19 over-expression may induce the elevated expression of mtp53 and interact with mtp53, leading to LAC progression. In addition, the high expression of mtp53 R175H is associated with poor overall survival inpatients. The simultaneous inhibition of H19 and mtp53 may provide a novel strategy for the effective control of LAC clinically.

The C-terminus of Gain-of-Function Mutant p53 R273H Is Required for Association with PARP1 and Poly-ADP-Ribose.

The C-terminal region of mtp53 confers GOF activity that mediates mtp53-PARP1 and PAR interactions assisting DNA replication, thus implicating new biomarkers for PARP inhibitor therapy.

Abstract 1826: Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers

Abstract Background: Disulfiram (DSF) in combination with copper pills has emerged as a safe and exciting anticancer strategy and is undergoing clinical trials in multiple human cancers. However, DSF breaks down in the gut or blood immediately and chances of cupric diethyldithiocarbamate (Cu-DDC), the active cytotoxic principle being formed at tumor sites is very low. Therefore, we have found that synthetic Cu-DDC is directly and potently cytotoxic against p53 mutant cancers. We report here for the first time the differential effects of Cu-DDC in robustly eliminating the mutant p53 proteins from tumor cells in contrast to its augmentation of the wild-type p53 protein and activation. Destabilization of Gain Of Function (GOF) p53 mutant proteins for overcoming oncogenic addiction and improving the therapeutic efficacy has been a long-sought, but unmet anticancer avenue. Methods: A panel of 7 P53 mutant cell lines (R248W, R273H,R173H), and p53 wt tumor and normal cells (HPBLs, dental pulp stem cells, IMR90 and HUVEC) were used. Immunoprecipitation, Western blotting, Annexin-V apoptotic assays, flow cytometry, immunocytochemistry were used to quantitate p53 protein. Mia-Paca2 xenografts developed in nude mice were administered Cu-DDC/BSA. Results: Cu-DDC generated peroxides but not superoxides in tumor cells. The GOF mutant p53 proteins, R248W and R273H in three different tumor cell lines were eliminated after Cu-DDC treatment in a time- and concentration-dependent manner. The loss of mutant p53 proteins was confirmed by three different procedures. The degradation occurred rapidly in 8-10 h at 100 nM Cu-DDC and was inhibited by proteasome blockers. The expression of the GRO-α oncogene, positively regulated by GOF R248W was decreased in Cu-DDC treated Mia-Paca2 cells. Cellular proteasome activity was not inhibited by Cu-DDC. Ubiquitination of mutant p53 proteins was not altered by Cu-DDC. In contrast, the wild-type p53 present in HCT116 and MCF-7 cells, and normal cell counterparts (HPBL, IMR-90 lung cells) was significantly upregulated and activated with enhanced p21cip1 expression. Cu-DDC triggered strong apoptosis irrespective of the p53 status, although the mutant p53 containing cells were 2 to 3-fold more sensitive in cytotoxicity assays (IC50 range 0.25-0.66 µM). A significant tumor growth delay accompanied by loss of mutant p53 protein was observed in MiaPaca2 xenografts. Conclusions: In moving the DSF anticancer repurposing forward, we describe the unique ability of Cu-DDC to rapidly eliminate oncogenic p53 mutant proteins and confer potent antitumor efficacy. The degradation was unrelated to the proteostasis involving the inactivation of NPL4 by Cu-DDC described previously (supported by a Cancer Prevention and Research Institute of Texas [CPRIT] grant RP170207 to KSS). Citation Format: Viswanath Arutla, Kalkunte S. Srivenugopal. Rapid and specific degradation of mutant p53 proteins by cupric diethyldithiocarbamate, the active cytotoxic component of disulfiram-copper repurposing in human cancers [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 1826.

Abstract 6034: Characterization of a novel syngeneic cSCC mouse model to advance immunotherapy treatments

Abstract Cutaneous Squamous Cell Carcinoma (cSCC) is the second most common malignancy diagnosed in Caucasians with an incidence of about one million cases per year. TP53 and p16 are the most common mutated genes in cSCC. Previous studies of cSCC have shown that patients with both p53 and p16 mutations have lower survival rates. There are currently no syngeneic mouse preclinical models to accelerate the efficacy of immunotherapy combinations to treat cSCC. Our laboratory has generated several genetically engineered mouse models (GEMMs) containing tissue-specific p53 and p16 gene mutations. Recently, we have immortalized three murine cSCC cell lines (cSCC-5690, cSCC-8006, and cSCC-8271) derived from spontaneous skin tumors from GEMMs. Therefore, we hypothesized that the double mutant cell line cSCC-5690 enhances cell survival, tumor aggressiveness, and has lower sensitivity to antibody immunotherapy. Protein and RNA expression of the cell lines were validated by western blotting and qRT-PCR assays. Sphere and soft agar assays were used to evaluate the stem cell population and tumorigenicity of the cell lines. Then, we examined the chemosensitivity with cisplatin through clonogenic assays. To further study the tumorigenicity of cells, we evaluated in vivo tumor growth on C57BL/6J immunocompetent mice by injecting subcutaneously the tumor cell lines. The three cell lines expressed different levels of keratin 14 (K14), but cSCC 5690 and 8006 expressed high levels of genes related to oncogenic pathways as well as mesenchymal markers. The cell line cSCC-5690 expressing mutant p53 R172H with p16 gene deletion, showed an enhanced sphere, and colony tumor formation and both tumorigenic cell lines (cSCC-5690, 8006) showed different sensitivities to cisplatin treatment. A higher tumor growth rate was observed with cSCC-5690 compared to cSCC-8006, while cSCC-8271 showed no tumor growth. Immunohistochemistry studies demonstrated that both cSCC-5690 and 8006 tumors expressed vimentin but only cSCC-5690 expressed K14. The most aggressive syngeneic tumor model (cSCC-5690) was used to evaluate the anti-tumor efficiency of anti-PD1, anti-CD47 antibodies, and STING-agonist. Preliminary results revealed a significant tumor response only to STING agonist (c-di-GMP) which activates the innate and adaptive immune response. Therefore, we were able to characterize all three cell lines and established a new mouse model of cSCC as a relevant platform for immunotherapy studies. Citation Format: Alanis Enid Rodriguez Rosario, Roberto Rangel, Jeffrey Myers. Characterization of a novel syngeneic cSCC mouse model to advance immunotherapy treatments [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 6034.

Abstract 2332: Repurposing simvastatin to inhibit the mevalonate pathway as a therapeutic strategy to treat high-grade serous ovarian cancer

Abstract Introduction: Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer, and its five-year survival rate has not changed appreciably in decades. As such, there is an imperative need for new and innovative therapies. Our lab has previously discovered that metastatic ascites-derived tumor cells have acquired a gain-of-function p53 mutation following interaction with the ovarian microenvironment in our orthotopic murine model of EOC. This p53 mutation is associated with an upregulation of the mevalonate pathway, which is known for its vital role in producing cholesterol. Tumor cells upregulate metabolic pathways as a survival advantage to fuel their rapid growth and metastasis. Simvastatin, a statin therapy, specifically targets HMG-CoA reductase, the rate-limiting enzyme of this pathway, and inhibits its activity. As such, simvastatin may serve as a potential therapeutic opportunity for EOC. Gain-of-function p53 mutations in the epithelial cells of the distal fallopian tube is now widely accepted as the origin of high-grade serous ovarian cancer. Thus, the purpose of our study was to investigate the relationship between p53 status and the mevalonate pathway in oviductal epithelial (OVE) cells as well as evaluate the effect of simvastatin treatment on OVE cell viability. Methods/Results: OVE cells were isolated from the distal oviducts of FVB/N mice with a p53 wildtype status, and we either deleted the Trp53 gene or introduced a gain-of-function R175H p53 mutation using CRISPR/Cas9. Cell viability was measured following treatment with varying doses of simvastatin and p53 mutant cells showed the greatest sensitivity (IC50: 5.48 uM) compared to p53 wildtype (IC50: 14.80 uM) and p53 knockout (IC50:9.78 uM) cells. For subsequent experiments, OVE cells were treated with 10 uM simvastatin or DMSO and subjected to Resazurin, Transwell migration, CyQUANT proliferative and Caspase-Glo 3/7 assays. Simvastatin significantly decreased resazurin reduction to resorufin in a time-dependent manner over 48 hours, regardless of p53 status, demonstrating reduced metabolic activity. Additionally, OVE cell invasion was significantly reduced in simvastatin treated p53 mutant cells, compared to p53 wildtype or p53 knockout cells. Simvastatin treatment diminished cell proliferation and enhanced apoptotic activity in all simvastatin treated OVE cells. Conclusion: Overall, simvastatin appears to inhibit tumorigenic processes in vitro. The next steps of this project are to test simvastatin’s effect on disease regression in a novel murine oviductal cell injection model of EOC recently developed by our lab. In light of these results, repurposing simvastatin as a therapy for ovarian cancer may significantly improve the way we treat this vicious disease. Citation Format: Madison Pereira, Kathy Matuszewska, Jacob Haagsma, Alice Glogova, Trevor G. Shepherd, Jim Petrik. Repurposing simvastatin to inhibit the mevalonate pathway as a therapeutic strategy to treat high-grade serous ovarian cancer [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 2332.

Identification of mutant p53-specific proteins interaction network using TurboID-based proximity labeling

BackgroundsAlthough several studies on mutant p53 reported cancer-promoting activities via “gain-of-function”, the mechanism underlying these differences in function between p53 R175H, R175P, and p53 wild-type (WT) remains unclear. MethodsLinking miniTurbo with p53 WT, R175H, and R175P, the expression of fusion and biotinylated proteins were assessed by Western blotting. The function and subcellular localization of fusion proteins were detected by apoptosis assay and immunofluorescence, respectively. Biotinylated proteins were analyzed by liquid chromatography-tandem mass spectrometry, followed by bioinformatics analysis. Small-scale pull-downs and Co-Immunoprecipitation were performed to validate the interaction between mutant or p53 WT and biotinylated proteins. ResultsThe fusion protein's cellular localization and function were consistent with those of previous studies on the corresponding p53. Comparative profiles of R175H versus WT showed that most of the interacting proteins belonged to the intracellular organelle lumen, and the pathways involved were metabolism and genetic information processing. Comparative profiles of R175P versus WT suggested that the majority of the interacting proteins belonged to the intracellular organelle lumen and the extracellular membrane-bounded organelle, and the pathways involved were metabolism and genetic information processing pathways. The comparison between R175H and R175P revealed that most interacting proteins belonged to the organelle lumen, and pathways involved were genetic information processing pathways. Finally, the mutation of p53 significantly altered the interaction with the target proteins were confirmed. ConclusionWe verified the reliability of the miniTurbo system and obtained candidate targets of mutant p53, which provided new thoughts on the mechanism of mutant p53 gain-of-function and new potential targets for cancer therapy.