P53 Wild-type Cells Research Articles

Enhanced Anti-Melanoma Activity of Nutlin-3a Delivered via Ethosomes: Targeting p53-Mediated Apoptosis in HT144 Cells.

This study evaluated ethosomes as a novel nanodelivery system for nutlin-3a, a known MDM2 inhibitor and activator of the p53 pathway, to improve nutlin-3a's poor solubility, limiting its bio-distribution and therapeutic efficacy. The potential of nutlin-3a-loaded ethosomes was investigated on two in vitro models of melanoma: the HT144 cell line p53wild-type and the SK-MEL-28 cell line p53mutated. Nutlin-3a-loaded ethosomes were characterized for their physicochemical properties and used to treat melanoma cells at different concentrations, considering nutlin-3a solution and empty ethosomes as controls. The biological effects on cells were evaluated 24 and 48 h after treatment by analyzing the cell morphology and viability, cell cycle, and apoptosis rate using flow cytometry and the p53 pathway's activation via Western blotting. The results indicate that ethosomes are delivery systems able to maintain nutlin-3a's functionality and specific biological action, as evidenced by the molecular activation of the p53 pathway and the biological events leading to cell cycle block and apoptosis in p53wild-type cells. Nutlin-3a-loaded ethosomes induced morphological changes in the HT144 cell line, with evident apoptotic cells and a reduction in the number of viable cells of over 80%. Furthermore, nutlin-3a-loaded ethosomes successfully modulated two p53-regulated proteins involved in survival/apoptosis, with up to a 2.5-fold increase in membrane TRAIL-R2 and up to an 8.2-fold decrease in Notch-1 (Notch intracellular domain, NICD) protein expression. The expression of these molecules is known to be altered or dysfunctional in a large percentage of melanoma tumors. Notably, ethosomes, regardless of their nutlin-3a loading, exhibited the ability to reduce HT144 melanoma cellular migration, as assessed in real time using xCELLigence, likely due to the modification of lipid rafts, suggesting their potential antimetastatic properties. Overall, nutlin-3a delivery using ethosomes appears to be a significantly effective means for upregulating the p53 pathway and downregulating active Notch-1, while also taking advantage of their unexpected ability to reduce cellular migration. The findings of this study could pave the way for the development of specific nutlin-3a-loaded ethosome-based medicinal products for cutaneous use.

P53 Status Influences the Anti-proliferative Effect Induced by IFITM1 Inhibition in Estrogen Receptor-positive Breast Cancer Cells.

Interferon-induced trans-membrane protein 1 (IFITM1) is known to be involved in breast cancer progression. We aimed to investigate its role in estrogen receptor (ER)-positive breast cancer cells with wild-type p53 and tamoxifen-resistant breast cancer cells. The ER-positive breast cancer cell lines, MCF-7 with wild-type p53 and T47D with mutant p53, were used. We established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with 4-hydroxytamoxifen. IFITM1 inhibition in MCF-7 cells significantly decreased cell growth and migration. MCF-7 cells with suppression of IFITM1 using siRNA or ruxolitinib showed reduced cell viability after tamoxifen treatment compared with that in the control MCF-7 cells. Unexpectedly, mRNA and protein levels of IFITM1 were decreased in TamR cells compared with those in MCF-7 cells. TamR cells with suppression of IFITM1 using siRNA or ruxolitinib showed no change in cell viability after treatment with tamoxifen. P53 knockdown using siRNA reduced the mRNA levels of IRF9 and increased mRNA and protein levels of SOCS3 in MCF-7 cells, suggesting that loss or mutation of p53 can affect the induction of IFITM1 via the JAK/STAT signaling pathway in breast cancer. Furthermore, MCF-7 cells with p53 knockdown using siRNA showed no decrease in cell viability after tamoxifen treatment or IFITM1 inhibition, indicating that p53 status may be important for cell death after tamoxifen treatment or IFITM1 inhibition. IFITM1 inhibition may enhance the sensitivity to tamoxifen based on p53-dependent enhancement of IFN signaling in wild-type p53, ER-positive breast cancer cells.

Exploring the anti-EBV potential of suberoylanilide hydroxamic acid: Induction of apoptosis in infected cells through suppressing BART gene expression and inducing lytic infection

Epstein-Barr virus (EBV) is linked to lymphoma and epithelioma but lacks drugs specifically targeting EBV-positive tumors. BamHI A Rightward Transcript (BART) miRNAs are expressed in all EBV-positive tumors, suppressing both lytic infection and host cell apoptosis. We identified suberoylanilide hydroxamic acid (SAHA), an inhibitor of histone deacetylase enzymes, as an agent that suppresses BART promoter activity and transcription of BART miRNAs. SAHA treatment demonstrated a more pronounced inhibition of cell proliferation in EBV-positive cells compared to EBV-negative cells, affecting both p53 wild-type and mutant gastric epithelial cells. SAHA treatment enhanced lytic infection in wild-type EBV-infected cells, while also enhancing cell death in BZLF1-deficient EBV-infected cells. It reduced BART gene expression by 85% and increased the expression of proapoptotic factors targeted by BART miRNAs. These findings suggest that SAHA not only induces lytic infection but also leads to cell death by suppressing BART miRNA transcription and promoting the apoptotic program.

DIPG-69. IN-VITRO DETERMINATION OF 5-AMINOLEVULINIC-ACID-TREATMENT EFFICACY IN PATIENT-DERIVED DIPG MODELS

Abstract BACKGROUND Diffuse Intrinsic Pontine Glioma (DIPG) presents a significant clinical challenge due to its inoperable nature and poor response to current treatments. Sonodynamic Therapy (SDT) is a non-invasive therapy that combines low-intensity ultrasound stimulation with a sonosensitizing agent. 5-Aminolevulinic Acid (5-ALA) as a sonosensitizer has yielded positive outcomes in in-vivo glioblastoma models, and a clinical trial (NCT05123534) for DIPG patients is currently ongoing. 5-ALA is a precursor of Protoporphyrin IX (PpIX), which preferentially accumulates in cancer cells and generates cytotoxic reactive oxygen species upon activation by ultrasound or light. Our work is aimed at investigating the accumulation of PpIX in in-vitro DIPG patient-derived cell models to predict the efficacy of 5-ALA SDT treatment. METHODS Patient derived DIPG cell lines (H3K27M, P53 mutant and wild type) were used to create three-dimensional spheroids. The spheroids were dosed with 5-ALA for 4 hours, and PpIX accumulation was quantified by measuring its fluorescence intensity within the cells. The spheroids were then exposed to controlled light stimulation as an in-vitro proxy for SDT. RESULTS We confirmed the uptake of 5-ALA and subsequent PpIX accumulation in all patient-derived DIPG spheroids. Light exposure of 5-ALA treated DIPG spheroids resulted in reduced viability of the tumor models, while 5-ALA itself did not show cytotoxic effects. The extent of PpIX accumulation and cytotoxicity upon PpIX light activation varied among DIPG models with higher sensitivity of p53 wild type cells. Further studies incorporating a larger number of DMG cells with p53 alterations are underway. CONCLUSIONS These findings indicate that SDT using 5-ALA as sonosensitizer may offer clinical benefits for treatment of DIPG, however treatment efficacy could vary considerably between patients. In vitro evaluation of PpIX accumulation and sensitivity to activation could potentially provide a means to stratify patients and to identify those most likely to benefit from 5-ALA-mediated SDT.

Abstract LB048: Unleashing the power of p53 mRNA therapeutics to suppress proliferation of p53 mutant tumors

Abstract The p53 protein, recognized as the guardian of the genome, plays a pivotal role in suppressing tumor formation. The TP53 gene, which encodes the p53 protein, is commonly subject to mutations and deletions in many cancer types resulting in loss of tumor suppressor activity. A significant proportion of patients diagnosed with lung, pancreatic, ovarian, and head and neck cancers exhibit the expression of mutant p53 in their tumors, correlating with unfavorable prognosis and survival outcomes.This study endeavors to assess the efficacy of therapeutic tumor suppressive mRNA which induces expression of wild-type p53 protein across diverse cancer types. Optimization of the 5’ and 3’ untranslated regions (UTRs) and the 5’ cap has been undertaken to enhance the efficient expression of p53 mRNA. The growth inhibitory potential of p53 mRNA therapy has been evaluated across p53 mutant cancer cell lines representing various cancer origins, along with normal or p53 wild-type cancer cell lines. We have substantiated that the p53 mRNA therapeutics can selectively inhibit the proliferation of p53 mutant cancer cells. Furthermore, we have confirmed that p53 mRNA treatment upregulated modulator of apoptosis (PUMA) and B-cell translocation gene 2 (BTG2) and cell cycle arrest via p21 upregulation. In vivo efficacy assessments of the lipid nanoparticle (LNP)-encapsulated p53 mRNA have been conducted in lung, pancreatic, and ovarian cancer xenograft models. Both regional intratumoral (i.t) and systemic intravenous (i.v) administrations of the p53 mRNA therapeutics have shown substantial suppression of tumor growth, leading to prolonged mouse survival. These results underscore the potential of the p53 mRNA therapeutics as a promising avenue for targeted cancer treatment, offering insights into the selective inhibition of p53 mutant cancer cells. Citation Format: Yong Ho Heo, Seung-Hyun Shin, Youngjin Han, Chang Gyu Lim, Innah Kim, Ji Hee Lee, Gunwoo Lee, Seongju Jeong, Yunjae Kim, Jooyun Byun, Daejin Kim, In Young Choi. Unleashing the power of p53 mRNA therapeutics to suppress proliferation of p53 mutant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB048.

Abstract 6588: Discovery of ASTX295, a potent, next-generation small molecule antagonist of MDM2 with differentiated pharmacokinetic profile

Abstract In response to cellular stress, the tumor suppressor p53 is activated to modulate cell cycle progression, DNA repair, and apoptosis. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 prevents its degradation and can reactivate p53 to elicit an anti-cancer effect. Targeting the p53-MDM2 interaction therefore remains a promising strategy for cancer therapy. However, development of first generation MDM2 antagonists has been challenged by dose-limiting, on-target bone marrow toxicities. Understanding of differential effects of p53 pathway activation in normal hematopoietic versus cancer cells (to be presented in a separate abstract) together with our expertise in structure-based drug design have led to the discovery of ASTX295, a potent MDM2 antagonist with differentiated pharmacokinetic profile aimed at sparing bone marrow toxicities and increasing the therapeutic index. Here, we present the first disclosure of the structure and pre-clinical characterisation of ASTX295. ASTX295 exhibits potent activity (IC50<1 nM) against MDM2 in an ELISA-based in vitro assay and induces significant growth reduction in p53 wild-type, MDM2-amplified SJSA-1 cells (GI50=27 nM). Antiproliferative activity of ASTX295 was further demonstrated in a panel of 219 p53 wild-type cell lines, with 143 cell lines showing GI50 values less than 1 μM and 50 showing values less than 0.1 μM. Effects of ASTX295 are shown in cell lines carrying functional p53 as confirmed in three p53 wild-type and mutant cell line pairs (SJSA1 and SN40R2, A2780 and A2780CP, HCT116 and HCT116 p53−/−). In addition to inhibiting cell cycle progression and cell proliferation, ASTX295 also potently induces apoptosis following 24-48 hour treatment. Further in vitro analyses of ASTX295 demonstrated an increase in the levels of p53 (EC50=10 nM after 2 hours) and its transcriptional targets such as p21 and MDM2. In vivo, ASTX295 shows robust induction of p53 and its target genes at 3 and 6 hours after oral administration together with dose-dependent inhibition of tumour growth in the SJSA-1 xenograft model. Importantly, ASTX295 exhibits optimised pharmacokinetic and pharmacodynamic profiles with relatively short duration of pathway modulation and a desired predicted human half-life of 2-8 hours. Based on our pre-clinical hypothesis on differential time-dependent sensitivities of normal versus cancer cells to p53 activation, achieving such a profile while maintaining potency may increase the therapeutic index. These data highlight the therapeutic potential of ASTX295, which is currently being tested in a Phase 1/2 clinical trial in advanced solid tumors with wild-type p53 (NCT03975387). We plan to present preliminary clinical data in a separate abstract at this meeting. Citation Format: Maria Ahn, Luke Bevan, Ildiko Buck, Celine Cano, Juan Castro, Ben Cons, Jane Endicott, Lynsey Fazal, Nicola Ferrari, Ian Hardcastle, Keisha Hearn, Rhian Holvey, Steven Howard, Chris Johnson, Claire Jennings, Justyna Kucia-Tran, Suzanne Kyle, John Lunec, John Lyons, Duncan Miller, David Rees, Martin Noble, David R. Newell, Judith Reeks, Harpreet Saini, Jeffrey St. Denis, Emiliano Tamanini, Huw Thomas, Neil Thompson, Mladen Vinkovic, George Ward, Nicola Wallis, Hugh Walton, Stephen Wedge, Pamela Williams, Elaine Willmore, Nicola Wilshire, Yan Zhao, Gianni Chessari. Discovery of ASTX295, a potent, next-generation small molecule antagonist of MDM2 with differentiated pharmacokinetic profile [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 6588.

Abstract 7590: Novel therapeutic approach for targeting p53 mutant colorectal cancers by affecting post-replicative DNA repair

Abstract Colorectal Cancer (CRC) is the third most common cause of cancer mortality in the US. A major problem in CRC management is relapse and progression to metastatic disease leading to poor overall survival. The tumor suppressor p53 is mutated in most CRCs and often drives metastasis and therapy resistance. Current therapeutic options targeting p53 mutant cancers show poor efficacy and frequently exhibit high toxicity. To address this clinical problem, we developed a novel therapeutic strategy for selective targeting of p53 mutant cancers. The strategy combines a thymidine analogue (e.g., trifluorthymidine/TFT, a component of TAS102) and poly (ADP-ribose) polymerase inhibitor (PARPi). We observed that TAS102 does not block DNA replication, but rather prompts post-replicative base-excision DNA repair (BER), and PARPi increases double-strand DNA breaks (DSBs) in p53 mutant cancer cells. Normal p53 wild type (p53wt) cells are arrested in the G1 phase and repair DNA. Thus, the TAS102-PARPi combination selectively targets p53-mutant cancer cells, resulting in the accumulation of DSBs and cell death. This novel strategy was examined in p53wt and p53-mutant tumor cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models. The drug combination was significantly more effective in inhibiting p53 mutant tumor growth and in prolonging survival compared to monotherapies. This strategy is currently being tested in a first-in-human dose-escalation Phase I study (NCT04511039) in patients with advanced CRC. Further investigation revealed that the TAS102-PARPi combination induced DNA damage response (DDR) and the G2 checkpoint, leading to G2 arrest in p53 mutant cancer cells. We observed that TAS102-PARPi induced phosphorylation of CDC2 (also known as CDK1) but did not increase phosphorylation of histone H3 (a marker of mitosis), therefore indicating activation of the G2 checkpoint. Furthermore, the roles of ATM and ATR kinases in the DDR and G2 responses were clarified using highly specific kinase inhibitors. The analysis of the DDR/G2 signaling response in isogenic p53wt and mutant CRC cell lines showed that inhibition of WEE1 kinase can selectively abrogate the G2 checkpoint in p53-mutant cells, resulting in marked DNA damage and cell death. Importantly, the blockade of WEE1 strongly synergized with the TAS102-PARPi combination, further enhancing its potency and efficacy against p53-mutant cancer cells. In summary, our research provides a foundation for a novel therapeutic strategy for p53 mutant cancers that may provide a paradigm shift in the treatment of these deadly cancers, increasing efficacy while minimizing toxic adverse effects. Citation Format: Mohammed M. Alruwaili, Natsumi Naranjo, Priyanka Rajan, Kyeong Beom Jo, Dae-Kyum Kim, Thomas Melendy, Robert Straubinger, Christos Fountzilas, Andrei Bakin. Novel therapeutic approach for targeting p53 mutant colorectal cancers by affecting post-replicative DNA repair [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 7590.

Abstract 4526: Inhibition of RNA Pol I transcription and induction of nucleolar stress in cancer cells treated with the experimental alpha-adrenergic receptor antagonist JP-1302

Abstract The inhibition of ribosome biogenesis through pharmacological means has drawn attention as a potential strategy in cancer therapy. In our previous study we highlighted a novel property of the FDA-approved antimalarial drug amodiaquine, demonstrating its ability to dose-dependently inhibit ribosomal RNA (rRNA) transcription, a critical step in ribosome biogenesis (Espinoza JA, et al Cell Death Diff, 2020). Amodiaquine was found to induce the degradation of the catalytic subunit of RNA polymerase I, known as POLR1A (RPA194), and resulting in nucleolar stress and the stabilization of p53. Notably, RNA Pol I shutdown occurred independently of DNA damage. Motivated by these findings, we sought to find additional inhibitors of ribosome biogenesis. Acridine derivatives display anti-cancer properties. The activity of acridines is mainly attributed to the planarity of these structures, which can intercalate within the DNA structure, and inhibition of chromatin associated protein targets including topoisomerases. JP-1302 is an experimental drug, on a 9-aminoacridine structural scaffold, acting as a selective alpha-2 adrenergic receptor antagonist. It was recently shown that JP-1302 inhibits transcription, blocking RNA Pol II phosphorylation and inhibiting p21 expression at a concentration of 10 μM (Mitchell DC, et al Nat Biotech, 2023). Given JP-1302's structural similarity to other acridine derivatives such as aminacrine, ethacridine, and the experimental compound BMH-21, all known to inhibit RNA Pol I transcription, we hypothesized that JP-1302 might share similar properties. The purpose of our new study was to evaluate JP-1302´s effect on ribosome biogenesis, p53, and cancer cell growth. We show here that JP-1302 acts as an inhibitor of RNA Pol I transcription. Treatment with JP-1302 induced a rapid decrease in the levels of POLR1A, cessation of rRNA synthesis and nucleolar stress already within six hours and at a concentration of 0.5 μM. JP-1302 treatment stabilized wild type p53 and enhanced p21 expression in the osteosarcoma line U2OS. RNA Pol I shutdown occurred in the absence of DNA damage response, as determined by gH2A.X staining. JP-1302 impaired the growth of both p53 wild type and p53 null HCT116 cells with GI50 values of around 0.6 μM, as well as in U2OS cells at 0.8 μM, as determined after 48 hours. Paradoxically, at very low concentrations JP-1302 appeared to transiently stimulate RNA synthesis, a phenomenon that will require further investigation. In conclusion, we identified JP-1302 as a novel inhibitor of ribosomal RNA synthesis, exerting this effect at concentrations that do not induce DNA damage, and independently of p53. Given that JP-1302 passes the blood-brain barrier in rodents, and accumulating in different brain regions, it is of future interest to evaluate this drug in brain tumor models. Citation Format: Sheetanshu Saproo, Styliani Papadaki, Jaime A. Espinoza, Jiri Bartek, Mikael S. Lindström. Inhibition of RNA Pol I transcription and induction of nucleolar stress in cancer cells treated with the experimental alpha-adrenergic receptor antagonist JP-1302 [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 4526.

Abstract 5988: The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma

Abstract Dysregulation of the Ubiquitin Proteasome System has been linked to many human diseases, including cancer. Expression of UBE4B ubiquitin ligase is associated with neuroblastoma patient outcomes and its functional roles in neuroblastoma pathogenesis are not known. We have recently identified a ubiquitin complex ITCH/UBE4B which mediates ubiquitination of Ku70 and c-FLIPL proteins for proteasomal degradation allowing HDAC inhibitor-mediated caspase-8 dependent apoptosis. We also confirmed that the deubiquitinase USP7, critical player in tumor suppression and DNA repair, can be a potential therapeutic target for neuroblastoma. Highly selective USP7 inhibitors have demonstrated significant antitumor activity in preclinical models of adult cancer, and we reported that these inhibitors alone can be more effective against neuroblastoma tumor growth. Our hypothesis is that USP7 inhibition will be more effective against neuroblastoma tumors through destabilization of ITCH/UBE4B complex protein targets allowing a stronger induction of cell death in response to treatment for the children. To evaluate efficacy of USP7 inhibitors in combination with HDAC inhibitors or chemotherapy against neuroblastoma tumor growth, neuroblastoma cell lines depleted for UBE4B or USP7 were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and apoptosis by caspase cleavage and PARP cleavage detection by western blot. We have evaluated also, whether ubiquitination/deubiquitination and degradation rates of p53, Ku70 and c-FLIPL proteins could modulate induction of apoptosis and necroptosis. USP7 inhibition resulted in decreases in cell viability in p53 wild-type neuroblastoma cell lines. USP7 inhibition induced apoptosis and necroptosis by increasing phosphorylation of MLKL, RIPK3 and RIPK1. In UBE4B or USP7 depleted cells, a huge induction of necroptosis and a small rate of apoptosis were observed. We also identified USP7 as a deubiquitinase of Ku70, stabilizing Ku70 at the basal level, leading to stabilization of Ku70/c-FLIPL/Bax complex. USP7 inhibition destabilizes ku70 and c-FLIPL for protein degradation leading to induction of apoptosis as well as necroptosis. UBE4B depletion in neuroblastoma inhibits HDAC inhibitors mediated caspase-8 mediated apoptosis but enhances necroptosis, due to the inhibition of Ku70 and c-FLIPL proteins degradation leading to the blockade of caspase-8 activation and the activation of the necroptosome. Our data suggests that USP7 and ITCH/UBE4B can control the switch between apoptosis and necroptosis in response to USP7 and HDAC inhibitors. USP7 inhibition and ITCH/UBE4B activation by HDAC inhibitors could be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, CARLA SAMPAIO, PETER E. ZAGE. The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma [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 5988.

Abstract 667: Identification of biomarkers of response to MDM2 inhibition in solid tumours using computational, multi-omics approaches

Abstract Background High-throughput drug screening and computational methods to associate genomic features of cell lines to drug sensitivity are valuable tools for predicting biomarkers of sensitivity. In addition, integrating genomic features to expression-based patterns could improve biomarker prediction and patient stratification. Particularly weighted gene co-expression networks represent an effective approach to identify key modules and possible biological mechanisms of sensitivity. Using a combination of cell panel screening and gene co-expression networks, we predicted markers of sensitivity to ASTX295 (MDM2i) in cancer cell lines and confirmed expression-based signatures in publicly available clinical datasets. Method ASTX295 was screened in a panel of 210 p53 wild-type cancer cell lines derived from a range of tumor tissues. ANOVA was used to identify significant associations of genomics features of cell lines to drug response. Transcriptomics profiling of apoptotic and non-apoptotic patient-derived cell lines was performed and significant differentially expressed genes were identified. Further, pathway enrichment and expression-based signatures identified in cell lines were further confirmed in TCGA patient data using weighted gene co-expression network analysis (WGCNA). Expression modules identified by WGCNA were correlated to genomic features and clinical parameters to identify potentially clinically relevant biomarkers. Results Analysis of the cell panel data identified CDKN2A loss as a statistically significant biomarker predictive of sensitivity to ASTX295. As mesothelioma is one of the indications with high prevalence of loss of CDKN2A, the anti-proliferative activity of ASTX295 was further confirmed in an independent panel of p53 wild-type, patient-derived mesothelioma cell lines. Further, pathway and transcriptional regulator analysis identified the Interferon signalling as significantly enriched in apoptotic cell lines and was confirmed in a TCGA mesothelioma patient data set and the module was found to be significantly correlated with a subgroup of P53 wild-type patients with CDKN2A loss. In conclusion, combining cell panel drug screening with co-expression networks helped to identify biomarkers associated with ASTX295 sensitivity, and provided new insights into the underlying mechanism of ASTX295 response. Citation Format: Harpreet Kaur Saini, Maria Ahn, George Ward, Justyna Kucia-Tran, Christina Gewinner, Nicola Ferrari, Jessica Brothwood, Luke Bevan, Matthew Davis, Lynsey Fazal, Martin Sims, Marc O'Reilly, Gianni Chessari, Roberta Ferraldeschi, John Lyons, Nicola Wallis, Neil Thompson. Identification of biomarkers of response to MDM2 inhibition in solid tumours using computational, multi-omics approaches [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 667.

Abstract 587: Tumor treating fields induce the integrated stress response, alter the transcriptional signatures of cellular metabolism, and modulate immune-related cytokines dependent and independent of p53

Abstract Tumor treating fields (TTFields) are an FDA-approved therapy for the treatment of glioblastoma and pleural mesothelioma. TTFields employ alternating electric fields delivered by cutaneous transducer arrays to induce cytostatic and cytotoxic effects on solid tumors. Additionally, recent studies suggest that TTFields may enhance anti-tumor immunity. We performed RNA-seq and multiplexed cytokine profiling in HCT116 wild-type and HCT116 p53-/- human colorectal cancer cells to elucidate TTFields’ effects on the transcriptome and secretome. TTFields lead to profound changes in the transcriptome related to metabolism, including downregulation of transcripts coding for multiple enzymes involved in the Krebs cycle, fatty acid synthesis, and glycolysis. Furthermore, though only HCT116 p53-wild type cells strongly induced the p53 pathway, both cell types’ transcriptomes showed strong downregulation of cell cycle progression. Our cytokine data showed that key tumor-promoting cytokine IL-8 was downregulated independently of p53. We identified upregulation of immunomodulatory cytokines MIF and MICB independently of p53. While MICB promotes NK cell activation, MIF drives tumor progression. Therefore, MICB may be one way in which TTFields enhance anti-tumor immunity; targeting MIF during TTFields treatment may lead to synergy by abolishing its pro-tumor effects. We also identified a p53-dependent decrease in the TRAIL decoy receptor DcR3, which normally protects tumor cells from TRAIL-induced apoptosis. Further mechanistic work showed that TTFields induce EIF2-alpha phosphorylation, the central protein involved in activating the integrated stress response (ISR), across multiple cancer types, leading to suppression of global protein translation. TTFields treatment also resulted in increased expression of surface calreticulin, an endoplasmic reticulum stress marker, which activates the ISR. Future mechanistic work will explore the impact of identified cytokines and the ISR on anti-tumor immunity both in vitro and in vivo, as well as metabolic alterations induced by TTFields. This work identifies potential biomarkers and rationales for therapeutic combinations exploiting TTFields-induced molecular alterations. Citation Format: Praveen R. Srinivasan, Andrew George, Charissa Chou, Maximilian Pinho-Schwermann, Maryam Ghandali, Vida Tajiknia, Yaara Porat, Moshe Giladi, Andrea Armstead, Alper Uzun, Eric T. Wong, Wafik S. El-Deiry. Tumor treating fields induce the integrated stress response, alter the transcriptional signatures of cellular metabolism, and modulate immune-related cytokines dependent and independent of p53 [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 587.

Vitamin C Inhibited Pulmonary Metastasis through Activating Nrf2/HO-1 Pathway.

As an important nutritional component, vitamin C (Vc) shows good antitumor activity in a variety of cancer, but there are few studies in pulmonary metastasis. In order to verify its anticancer and antimetastatic effect, the study sets up H22 pulmonary metastasis mouse model. The results show that intraperitoneal injection of Vc inhibits pulmonary metastasis through up-regulating the expression of Nrf2, HO-1, cleaved caspases 3 and 9, and causing DNA damage and apoptosis which is similar to the pro-oxidant effect of Vc in p53 null cells (H1299 cells). Meanwhile, oral administration of Vc up-regulates the expression of p53, directly activates Nrf2/HO-1 pathway, increases expression of cleaved caspases 3 and 9, and ultimately inhibits pulmonary metastasis, which is the same as the antioxidant result of Vc in p53 wild-type cells. In addition, Vc inhibits the proliferation and migration of lung cancer cells in a concentration-dependent manner and has little cytotoxic effects on normal cells. Notably, the experiment further illustrates that besides intravenous Vc, oral Vc significantly inhibits the pulmonary metastasis in mice. All in all, these findings provide new clues for Vc-treated pulmonary metastasis in clinical research.

NEAT1 repression by MED12 creates chemosensitivity in p53 wild-type breast cancer cells.

Breast cancer is often treated with chemotherapy. However, the development of chemoresistance results in treatment failure. Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been shown to contribute to chemoresistance in breast cancer cells. In studying the transcriptional regulation of NEAT1 using multi-omics approaches, we showed that NEAT1 is up-regulated by 5-fluorouracil in breast cancer cells with wild-type cellular tumor antigen p53 but not in mutant-p53-expressing breast cancer cells. The regulation of NEAT1 involves mediator complex subunit 12 (MED12)-mediated repression of histone acetylation marks at the promoter region of NEAT1. Knockdown of MED12 but not coactivator-associated arginine methyltransferase 1 (CARM1) induced histone acetylation at the NEAT1 promoter, leading to elevated NEAT1 mRNAs, resulting in a chemoresistant phenotype. The MED12-dependent regulation of NEAT1 differs between wild-type and mutant p53-expressing cells. MED12 depletion led to increased expression of NEAT1 in a wild-type p53 cell line, but decreased expression in a mutant p53 cell line. Chemoresistance caused by MED12 depletion can be partially rescued by NEAT1 knockdown in p53 wild-type cells. Collectively, our study reveals a novel mechanism of chemoresistance dependent on MED12 transcriptional regulation of NEAT1 in p53 wild-type breast cancer cells.

A synergistic two-drug therapy specifically targets a DNA repair dysregulation that occurs in p53-deficient colorectal and pancreatic cancers

The tumor-suppressor p53 is commonly inactivated in colorectal cancer and pancreatic ductal adenocarcinoma, but existing treatment options for p53-mutant (p53Mut) cancer are largely ineffective. Here, we report a therapeutic strategy for p53Mut tumors based on abnormalities in the DNA repair response. Investigation of DNA repair upon challenge with thymidine analogs reveals a dysregulation in DNA repair response in p53Mut cells that leads to accumulation of DNA breaks. Thymidine analogs do not interrupt DNA synthesis but induce DNA repair that involves a p53-dependent checkpoint. Inhibitors of poly(ADP-ribose) polymerase (PARPis) markedly enhance DNA double-strand breaks and cell death induced by thymidine analogs in p53Mut cells, whereas p53 wild-type cells respond with p53-dependent inhibition of the cell cycle. Combinations of trifluorothymidine and PARPi agents demonstrate superior anti-neoplastic activity in p53Mut cancer models. These findings support a two-drug combination strategy to improve outcomes for patients with p53Mut cancer.

Nanoencapsulation of MDM2 Inhibitor RG7388 and Class-I HDAC Inhibitor Entinostat Enhances their Therapeutic Potential Through Synergistic Antitumor Effects and Reduction of Systemic Toxicity.

Inhibitors of the p53-MDM2 interaction such as RG7388 have been developed to exploit latent tumor suppressive properties in p53 in 50% of tumors in which p53 is wild-type. However, these agents for the most part activate cell cycle arrest rather than death, and high doses in patients elicit on-target dose-limiting neutropenia. Recent work from our group indicates that combination of p53-MDM2 inhibitors with the class-I HDAC inhibitor Entinostat (which itself has dose-limiting toxicity issues) has the potential to significantly augment cell death in p53 wild-type colorectal cancer cells. We investigated whether coencapsulation of RG7388 and Entinostat within polymeric nanoparticles (NPs) could overcome efficacy and toxicity limitations of this drug combination. Combinations of RG7388 and Entinostat across a range of different molar ratios resulted in synergistic increases in cell death when delivered in both free drug and nanoencapsulated formats in all colorectal cell lines tested. Importantly, we also explored the in vivo impact of the drug combination on murine blood leukocytes, showing that the leukopenia induced by the free drugs could be significantly mitigated by nanoencapsulation. Taken together, this study demonstrates that formulating these agents within a single nanoparticle delivery platform may provide clinical utility beyond use as nonencapsulated agents.

Celecoxib enhances the response of tumor cells to cisplatin through upregulating PUMA in non–small cell lung cancer carrying wild-type p53

Abstract Celecoxib, a cyclooxygenase-2 inhibitor, can enhance the efficacy of chemotherapy; however, its effect seems inconsistent. In this study, we investigated whether celecoxib would increase the antiproliferative effects of cisplatin in human lung cancer cells. Our data demonstrated the synergistic effects of celecoxib with cisplatin in wild-type p53 cells and their antagonistic effects in mutated or deleted p53 cells. Combination indices of 0.82 to 0.93 reflected a synergistic effect between celecoxib and cisplatin in lung cancer cells with wild-type p53. Combination indices of 1.63 to 3.00 reflected antagonism between celecoxib and cisplatin in lung cancer cells with mutated or deleted p53. Compared with that in cells with mutated or deleted p53, apoptosis significantly increased with the addition of celecoxib and cisplatin in wild-type p53 cells (P < 0.05). Moreover, the results in vivo were similar to those in vitro: celecoxib combined with cisplatin slowed tumor growth in wild-type p53 groups and not in mutated or deleted p53 groups. In addition, celecoxib promoted p53 translocation into the nucleus and upregulated active p53 expression in wild-type p53 cells. Celecoxib combined with cisplatin upregulated PUMA (PUMA is a downstream gene of p53) after active p53 increased in wild-type p53 cells. In summary, the combination of celecoxib and cisplatin demonstrates clear synergistic effects in wild-type p53 cells and antagonistic effects in mutated or deleted p53 cells. The synergistic effect was achieved by apoptosis, induced by upregulating PUMA. Our results will provide a new treatment strategy for patients carrying wild-type p53, insensitive to cisplatin.

Aspirin boosts the synergistic effect of EGFR/p53 inhibitors on lung cancer cells by regulating AKT/mTOR and p53 pathways.

The regimen of afatinib and vinorelbine has been used to treat breast or lung cancer cells with some limitations. Aspirin alone or in combination with other agents has shown unique efficacy in the treatment of cancer. We designed a preclinical study to investigate whether the triple therapy of aspirin, afatinib, and vinorelbine could synergistically inhibit the growth of p53 wild-type nonsmall cell lung cancer (NSCLC) cells. Three NSCLC cells A549, H460, and H1975 were selected to study the effect of triple therapy on cell proliferation and apoptosis. Compared to single agents, triple therapy synergistically inhibited the proliferation of lung cancer cells with combination index <1. Meanwhile, the therapeutic indexof triple therapy was superior to that of single agents, indicating a balance between efficacy and safety in the combination of three agents. Mechanistic studies showed that triple therapy significantly induced apoptosis by decreasing mitochondrial membrane potential, increasing reactive oxygen species, and regulating mitochondria-related proteins. Moreover, epidermal growth factor receptor(EGFR) downstream signaling proteins including JNK, AKT, and mTOR were dramatically suppressed and p53 was substantially increased after NSCLC cells were exposed to the triple therapy. We provided evidence that the triple therapy of aspirin, afatinib and vinorelbine synergistically inhibited lung cancer cell growth through inactivation of the EGFR/AKT/mTOR pathway and accumulation of p53, providing a new treatment strategy for patients with p53 wild-type NSCLC.

Mutant p53 murine oviductal epithelial cells induce progression of high-grade serous carcinoma and are most sensitive to simvastatin therapy in vitro and in vivo

High-grade serous carcinoma (HGSC) is the most common and aggressive subtype of epithelial ovarian cancer, characterized by gain-of-function TP53 mutations originating in the fallopian tube epithelium. Therapeutic intervention occurs at advanced metastatic disease, due to challenges in early-stage diagnosis, with common disease recurrence and therapy resistance despite initial therapy success. The mevalonate pathway is exploited by many cancers and is potently inhibited by statin drugs. Statins have shown anti-cancer activity in many, but not all cancers. Here, we investigated the role of p53 status in relation to mevalonate pathway signaling in murine oviductal epithelial (OVE) cells and identified OVE cell sensitivity to statin inhibition. We found that p53R175H mutant and Trp53 knockout OVE cells have increased mevalonate pathway signaling compared to p53 wild-type OVE cells. Through orthotopic implantation to replicate the fallopian tube origin of HGSC, p53R175H mutant cells upregulated the mevalonate pathway to drive progression to advanced-stage ovarian cancer, and simvastatin treatment abrogated this effect. Additionally, simvastatin was more efficacious at inhibiting cell metabolic activity in OVE cells than atorvastatin, rosuvastatin and pravastatin. In vitro, simvastatin demonstrated potent effects on cell proliferation, apoptosis, invasion and migration in OVE cells regardless of p53 status. In vivo, simvastatin induced ovarian cancer disease regression through decreased primary ovarian tumor weight and increased apoptosis. Simvastatin also significantly increased cytoplasmic localization of HMG-CoA reductase in ovarian tumors. Downstream of the mevalonate pathway, simvastatin had no effect on YAP or small GTPase activity. This study suggests that simvastatin can induce anti-tumor effects and could be an important inhibitor of ovarian cancer progression.

Loss of p53 Impairs Death Receptor Expression and Confers Resistance to CD19-Directed CAR T-Cell Therapy in B-Cell Precursor Acute Lymphoblastic Leukemia

Background In recent years, new therapy strategies such as the antibody-drug conjugate inotuzumab ozogamicin, the bi-specific T-cell engager blinatumomab, and chimeric antigen receptor (CAR) T-cells have been introduced to salvage relapsed precursor B acute lymphoblastic leukemia (ALL). However, recent evidence indicates that these new therapies are ineffective in TP53-aberrant ALL, although the mechanism remains unclear. TP53 is the most frequently mutated gene in cancer, but in ALL, mutations or deletions affecting TP53 are rare, with an incidence of only 3% at diagnosis. However, at relapse, TP53 aberrations are found in approximately 12% of the patients and predict a very poor outcome. Consequently, relapsed TP53 deleted ALL is now classified as ‘very high risk’. Since p53-mediated apoptosis is an endpoint for many (cytotoxic) drugs, loss of p53 function induces therapy resistance to classical chemotherapeutics. Hence, there is a clear clinical need for more effective strategies to treat TP53 aberrant relapsed ALL. Methods Using CRISPR/Cas9, we introduced frameshift mutations in the TP53 gene to generate isogenic wild type and knockout models in TP53 WT ALL cell lines Nalm6 and RCH-ACV. Additionally, we prepared CD19-directed CAR T-cells with a 4-1BB co-stimulatory domain from three healthy donors by isolating, transducing, and expanding CD4+ and CD8+ T-cells separately. With flow cytometry, we assessed survival of p53 WT and p53 KO leukemic cells in the presence of CD4+ and CD8+ CAR T-cells at a 1:1 CD4:CD8 ratio. Moreover, we performed mRNA profiling by qRT-PCR and cell membrane profiling by flow cytometry to determine how loss of p53 affects CAR T mediated killing. Initial findings in cell lines were then validated in patient-derived xenografts. Results We observed that loss of p53 led to up to a 35% decrease in killing by overnight CAR T treatment in both cell line models (Figure 1A). Although loss of p53 has been linked to reduced cell surface expression of CD19 (Pan et al., 2020, Leukemia), no changes in CD19 expression were observed in response to p53 loss. In search for an explanation for the diminished killing, we performed mRNA profiling and found a reduced expression of death receptors CD95/Fas and DR5/TRAILR2 in p53-deficient cells, which was confirmed at the protein level with flow cytometry (Figure 1B). Indeed, CRISPR/Cas9 mediated loss of CD95/Fas expression in p53 wild type cells reduced CAR T mediated cell killing, consistent with a key role for CD95/Fas in T-cell mediated killing. Conversely, treatment of p53 proficient cells with MDM2 inhibitors induced expression of CD95/Fas and DR5/TRAILR2 (Figure 1B), which translated into enhanced killing of target cells. Most of these observations have already been recapitulated in a diagnosis-relapse pair of patient-derived cells in which the diagnosis was p53wild typeand the relapse was p53 deficient (Figure 1A-B). We observed lower baseline mRNA expression of CD95/Fas and DR5/TRAILR2 in p53-deficient cells, accompanied by a 50% reduction in killing relative to the p53 wild type controls. Conversely, MDM2 inhibition increased death receptor expression only in p53-proficient cells. The ongoing experiments validating enhanced killing after MDM2 inhibition in patient-derived cells will be presented during the meeting. Conclusions Loss of p53 impairs expression of CD95/Fas and DR5/TRAILR2, thereby protecting ALL cells from CAR T induced cell death. In addition, our results indicate that the p53-controlled expression of these receptors may be exploited to increase the efficacy of CAR T-cell therapy in p53-proficient patients.

ROCK inhibition reduces the sensitivity of mutant p53 glioblastoma to genotoxic stress through a Rac1-driven ROS production.

Resistance to radio and chemotherapy in Glioblastoma (GBM) is correlated with its malignancy, invasiveness, and aggressiveness. The Rho GTPase pathway plays important roles in these processes, but its involvement in the GBM response to genotoxic treatments remains unsolved. Inhibition of this signaling pathway has emerged as a promising approach for the treatment of CNS injuries and diseases, proving to be a strong candidate for therapeutic approaches. To this end, Rho-associated kinases (ROCK), classic downstream effectors of small Rho GTPases, were targeted for pharmacological inhibition using Y-27632 in GBM cells, expressing the wild-type or mutated p53 gene, and exposed to genotoxic stress by gamma ionizing radiation (IR) or cisplatin (PT). The use of the ROCK inhibitor (ROCKi) had opposite effects in these cells: in cells expressing wild-type p53, ROCKi reduced survival and DNA repair capacity (reduction of γH2AX foci and accumulation of strand breaks) after stress promoted by IR or PT; in cells expressing the mutant p53 protein, both treatments promoted longer survival and more efficient DNA repair, responses further enhanced by ROCKi. The target DNA repair mechanisms of ROCK inhibition were, respectively, an attenuation of NHEJ and NER pathways in wild-type p53 cells, and a stimulation of HR and NER pathways in mutant p53 cells. These effects were accompanied by the formation of reactive oxygen species (ROS) induced by genotoxic stress only in mutant p53 cells but potentiated by ROCKi and reversed by p53 knockdown. The p53-dependent actions of ROCKi were completely abolished by N-acetyl-L-cysteine (NAC) treatment or Rac1 knockdown, as Rac-GTP levels were specifically elevated by ROCK inhibition only in mutant p53 cells. Combining IR or PT and ROCKi treatments broadens our understanding of the sensitivity and resistance of, respectively, GBM expressing wild-type or mutant p53 to genotoxic agents. Our proposal may be a determining factor to improve the efficiency and assertiveness of CNS antitumor therapies based on ROCK inhibitors. SIGNIFICANCE: The use of ROCK inhibitors in association with radio or chemotherapy modulates GBM resistance and sensitivity depending on the p53 activity, suggesting the potential value of this protein as therapeutic target for tumor pre-sensitization strategies.