Tumor Suppressor Protein Research Articles

Integrated mathematical and experimental modeling uncovers enhanced EMT plasticity upon loss of the DLC1 tumor suppressor.

Epithelial-mesenchymal transition (EMT) plays an essential role in embryonic development, wound healing, and tumor progression. Partial EMT states have been linked to metastatic dissemination and drug resistance. Several interconnected feedback loops at the RNA and protein levels control the transition between different cellular states. Using a combination of mathematical modeling and experimental analyses in the TGFβ-responsive breast epithelial MCF10A cell model, we identify a central role for the tumor suppressor protein Deleted in Liver Cancer 1 (DLC1) during EMT. By extending a previous model of EMT comprising key transcription factors and microRNAs, our work shows that DLC1 acts as a positive regulator of TGFβ-driven EMT, mainly by promoting SNAIL1 expression. Our model predictions indicate that DLC1 loss impairs EMT progression. Experimental analyses confirm this prediction and reveal the acquisition of a partial EMT phenotype in DLC1-depleted cells. Furthermore, our model results indicate a possible EMT reversion to partial or epithelial states upon DLC1 loss in MCF10A cells induced toward mesenchymal phenotypes. The increased EMT plasticity of cells lacking DLC1 may explain its importance as a tumor suppressor.

Circular Nucleic Acids Act as an Oncogenic MicroRNA Sponge to Inhibit Hepatocellular Carcinoma Progression

Background: Aberrant expression of microRNAs in neoplastic lesions may serve as potential personalized therapeutic targets. To inhibit oncogenic microRNAs (oncomiRs) expression and restore tumor suppressor proteins, linear miRNA sponges have been developed, leading to several drugs in clinical trials. Despite their efficacy, chemically synthesized miRNA inhibitors face challenges with sustained inhibition and high production costs, hindering widespread clinical adoption. Additionally, single-stranded circular RNAs (circRNAs) act as miRNA sponges, enhancing protein expression and demonstrating stability and therapeutic potential in cancer treatment. Our approach involves the use of synthetic single-stranded circular nucleic acids, including circDNA and circRNA, to selectively target and inhibit a variety of aberrantly overexpressed oncomiRs in tumors. The objective of this strategy is to restore the expression levels of multiple tumor suppressor factors and to suppress the malignant progression of tumors. Methods: Our methodology comprises a two-step process. First, we identified tumor suppressor genes (TSGs) with abnormally low expression in hepatocellular carcinoma (HCC) tumor cells by transcriptomic analysis and targeted the upstream cancer miRNA clusters of these TSGs. Second, we designed and validated a fully complementary circDNA or circRNA construct, ligated by T4 DNA ligase or T4 RNA ligase, respectively, that specifically targets the sponge oncomiRs both in vitro and in vivo to inhibit the malignant progression of HCC. Results: CircNAs demonstrated superior, long-lasting therapeutic efficacy against HCC compared to inhibitors. Furthermore, we compared the immune effects in vivo of three different nucleic acid adsorption carriers, including commercial miRNA inhibitor, circDNA, and circRNA. We found that the miRNA inhibitor activates a more robust inflammatory response compared to circDNA and circRNA. Conclusions: These findings underscore the substantial therapeutic potential of circDNA in tumorigenesis and provide novel insights for the formulation of personalized treatment plans for malignant tumors, such as HCC.

The Central Role of Ribosomal Proteins in p53 Regulation

The tumor suppressor protein p53 prevents the malignant transformation of cells by responding to DNA damage, oncogene activation, and abnormal growth signals including ribosome assembly defects. Under normal conditions, p53 activity is controlled by the regulatory proteins MDM2 and MDM4, which suppress its function through ubiquitin-mediated degradation and transcriptional inhibition. A subset of ribosomal proteins initiates the p53 response to impaired ribosome biogenesis. The ability of some ribosomal proteins to control MDM2 and MDM4 activities, and thereby p53, underscores an intriguing aspect of cell biology: proteins primarily known for their roles in ribosome function can exert extra-ribosomal functions. One notable example is the cellular RNA-protein complex involving RPL5, RPL11, and 5S rRNA (5S RNP) which inhibits MDM2 and stabilizes p53. Another RP, RPL22, is frequently mutated in cancers with microsatellite instability and its paralog RPL22L1 is often amplified. Recent studies have revealed that RPL22 directly modulates the alternative splicing of MDM4 to promote p53 activation, suggesting that the ribosomal protein-p53 relationship is more complex than previously thought. Cellular responses to ribosome biogenesis inhibition extend beyond general alterations in transcription and translation to actively determine cancer cell fate by selectively engaging tumor-suppressor pathways. RPL22’s effect on MDM4 and other mRNA splicing events is a striking example. A better understanding of the mechanisms involved could guide the development of improved cancer treatments.

Assessing phototoxic drug properties of hydrochlorothiazide using human skin biopsies

The diuretic drug hydrochlorothiazide (HCT) is associated with an increased risk of non-melanoma skin cancer upon UV exposure. The underlying cellular and molecular mechanisms behind this association remain elusive. Herein, a human skin model to assess the photocarcinogenic effects of HCT is established. Skin biopsies collected from human body donors are treated with HCT and irradiated with 300 mJ/cm2 low dose UVA or UVB or with 5 J/cm2 high dose UVA. In HCT-treated biopsies but not in control, low dose UVA irradiation results in activation and nuclear translocation of the tumor-suppressor protein p53 accompanied by an upregulated gene expression of p53-negative regulator MDM2. High dose UVA additionally provokes DNA damage and initiation of pro-inflammatory gene expression. In contrast, UVB induces pronounced DNA damage, p53 protein activation, gene expression of MDM2 and inflammatory marker genes in both HCT-treated biopsies and untreated control. In summary, in HCT-treated skin biopsies, activation of the p53-MDM2 axis, induction of DNA damage, and inflammatory response depends on UVA-dosage and may influence skin carcinogenesis over time. This human model eliminates the need for animal testing and mitigates species difference, offering a valuable tool for future drug development and safety testing.

Microtubule associated serine/threonine kinase-3 inhibits the malignant phenotype of breast cancer by promoting phosphorylation-mediated ubiquitination degradation of yes-associated protein

BackgroundMicrotubule associated series/threonine kinase-3 (MAST3) is a member of microtubule associated serine/threonine kinase family (MAST1-4, MAST-like), and the expression and underlying molecular mechanism of MAST3 in human tumors, including breast cancer, is not yet elucidated.MethodsWe employed immunohistochemistry to assess the significant expression of MAST3 in breast cancer tissue samples. Additionally, we utilized an overexpression vector and shRNA to bi-directionally regulate MAST3 expression, aiming to observe the impact of MAST3 on the proliferation, migration, and invasion capabilities of breast cancer cells. Furthermore, we employed immunoprecipitation, immunoblotting, luciferase reporter genes and real-time quantitative PCR to investigate the interaction between MAST3 and YAP, as well as the regulatory effects on the expression of Hippo pathway-related target genes.ResultsLow MAST3 expression was observed both in breast cancer cells and tissues, which was significantly associated with advanced tumor T stage, lymph node metastasis, and poor patient prognosis. Functional experiments found that overexpression of MAST3 can gradually inhibit the proliferation and invasion of breast cancer cells, knocking-out MAST3 showed the opposite functional effect. Immunoprecipitation showed that MAST3 interacts with the key effector factor, yes-associated protein (YAP), in the Hippo pathway. The combination of MAST3-YAP promoted the phosphorylation of YAP, which led to its degradation through the ubiquitin-proteasome pathway and reduced nuclear translocation.ConclusionsMAST3 was identified as a novel tumor suppressor protein in breast cancer, which directly regulates the expression of YAP through the non-dependent mammalian sterile-20-like (MST)-large tumor suppressor (LATS) classical signaling pathway, providing a theoretical and experimental basis for the development of small-molecule tumor inhibitors in breast cancer.

Stronger preference of human tumor suppressor protein BRCA1 for an open-planar Holliday junction: Insights from a combined spectroscopic and computational study.

Stronger preference of human tumor suppressor protein BRCA1 for an open-planar Holliday junction: Insights from a combined spectroscopic and computational study.

Therapeutic Potential of Translational Readthrough at Disease‐Associated Premature Termination Codons From Tumor Suppressor Genes

ABSTRACTTumor suppressor genes are frequently targeted by mutations introducing premature termination codons (PTC) in the protein coding sequence, both in sporadic cancers and in the germline of patients with cancer predisposition syndromes. These mutations have a high pathogenic impact since they generate C‐terminal truncated proteins with altered stability and function. In addition, PTC mutations trigger transcript degradation by nonsense‐mediated mRNA decay. Suppression of PTC by translational readthrough restores protein biosynthesis and stabilizes the PTC‐targeted mRNA, making a suitable therapeutic approach the reconstitution of active full‐length tumor suppressor proteins by pharmacologically‐induced translational readthrough. Here, we review the recent advances in small molecule pharmacological induction of translational readthrough of disease‐associated PTC from tumor suppressor genes, and discuss the therapeutic potential of translational readthrough in specific groups of patients with hereditary syndromic cancers.

Neutrophil membrane engineered human umbilical cord MSC-derived sEVs enhance anti-tumor efficacy for gastric cancer via delivering pentraxin 3.

Neutrophil membrane engineered human umbilical cord MSC-derived sEVs enhance anti-tumor efficacy for gastric cancer via delivering pentraxin 3.

Quercetin Mitigates Sulfasalazine-Induced Toxicity by Inhibiting p53-Dependent Apoptosis and DNA Damage in Rat Epididymis, Semen and Bone Marrow.

Sulfasalazine, an anti-inflammatory drug used in the treatment of inflammatory bowel disorders poses a threat to male reproductive potential. With a growing interest in the therapeutic ability of dietary flavonoids to mitigate drug-related reproductive dysfunction in males, we evaluated the possible protective potentials of quercetin against sulfasalazine-induced genotoxicity in the bone marrow and apoptosis in testis, semen, and epididymis of Wistar rats. Twenty rats were placed into four groups (n = 5) and orally administered the following for 14 consecutive days: Group 1: 0.5% sodium carboxymethyl cellulose (1 ml/kg). Group 2: quercetin (20 mg/kg). Group 3: sulfasalazine (600 mg/kg). Group 4: sulfasalazine (600 mg/kg) + quercetin (20 mg/kg). Tumor suppressor protein (Tp53), caspase 9, and caspase 3 levels were increased in semen, while TUNEL positive sperm increased in epididymis of sulfasalazine-treated rats, indicative of apoptotic death of sperm. These were significantly decreased in sulfasalazine-treated rats co-administered with quercetin. However, levels of Tp53, caspase 9 and 3 remained unchanged, while TUNEL-stained sperm cells were not evident in testes of rats treated with sulfasalazine. Again, sulfasalazine increased the frequency of micronuclei (MN) in bone marrow of treated rats, evident of its genotoxic potential. MN frequency was, however, decreased in their bone marrow on co-administration of quercetin. In conclusion, our findings suggest that quercetin holds promise in ameliorating the apoptotic death of sperm cells associated with sulfasalazine's detrimental effect on male fertility and DNA.

Abstract 3293: Exploiting PEGylation of CCN5 for tumor immunity and therapy of triple-negative breast cancer: A new and effective approach for TNBC therapy

Abstract Triple-negative breast cancer (TNBC) is defined by a lack of expressions of three receptors. These include estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor rector 2 (HER2). TNBC accounts for ∼15-17% of all breast cancers (BC) and is the most aggressive subtype of metastatic BC. TNBC incidence is high among BRCA1 mutation carriers, women with the age of 50 or below, and African American descent. The therapeutic opportunities for TNBC are limited to chemotherapy as targeted options are deficient, resulting in elevated risk of recurrence and mortality risk. Over the last five years, new strategies for treating TNBC have transformed significantly by introducing PD-1/PD-L1-immune checkpoint inhibitors (ICIs) and others. However, only eighteen-20% of PD-L1 positive TNBC patients with metastasis respond to ICIs with or without chemotherapy and enhance the antitumor response of therapeutic CD8+ T cells. The low overall response rate of ICIs in TNBC patients is due to insufficient PD-L1/PD-1 interaction blockade. Consequently, an unmet need exists to revolutionize treatment regimens by replacing or modifying them with ones that are more effective and less toxic. Previous studies demonstrate that CCN5 (also known as WISP-2), which is family of cellular communication Network (CCN) factors, is considered a tumor suppressor and anti-invasive protein as it promotes the growth arrest and progression of TNBC cells. Our previous studies showed that recombinant CCN5 (hrCCN5) therapy is weak for systemic delivery due to poor bioavailability. Thus, only intratumoral injection of CCN5 was considered and found effective with no toxicity in animal studies, but this is not an ideal therapeutic strategy for progressive BC therapy. To overcome this limitation, we implemented the protein PEGylation/PEG conjugation technique that increased the bioavailability and activities of CCN5 and reverses tumor growth and metastasis and increases prolonged overall survival in an MDA-MB-231tumor xenograft model. Further, we found that PEGCCN5 significantly increased CD8+ T cells decreased myeloid infiltration and PD-L1 expression in tumor cells. Thus, we consider PEGCCN5 can emerge as a new, beneficial immunotherapeutic drug for TNBC treatment alone or in combination with αPD-1, chemotherapy, or both. Citation Format: Sunil P. Upadhyay, Axel H. Breier, Inamul Haque, Archana De, Snigdha Banerjee, Mohiuddin Quadir, Sushanta K. Banerjee. Exploiting PEGylation of CCN5 for tumor immunity and therapy of triple-negative breast cancer: A new and effective approach for TNBC therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3293.

Abstract 6808: DOCK1 as a therapeutic target for EGFR resistance in cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is an aggressive cancer of the bile ducts, characterized by poor prognosis and limited treatment options due to late-stage diagnosis and resistance to targeted therapies. Mutations in critical pathways, including the Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR), drive CCA progression, while resistance mechanisms and mutation weaken the effectiveness of available therapies. Dedicator of Cytokinesis 1 (DOCK1), a guanine nucleotide exchange factor, has emerged as a key player in cancer progression, particularly in modulating cell signaling. In this study, we investigated the therapeutic potential of targeting DOCK1 in CCA via modulating EGFR signaling. Our assessment across five distinct patient cohorts demonstrates that DOCK1 is significantly overexpressed in CCA tumors compared to normal cholangiocytes, underscoring its pivotal role in promoting tumor growth and aggressiveness. Pharmacological inhibition of DOCK1 using the small-molecule inhibitors CPYPP and TBOPP, as well as siRNA-mediated knockdown, resulted in a marked reduction in HuCCT1 and EGI1 CCA cell lines proliferation and migration. This was accompanied by an increase in tumor suppressor proteins p53 and p21, along with a decrease in proliferation markers PCNA and cyclin D1. Moreover, siRNA-mediated reduction of DOCK1 expression significantly induced apoptosis and cytotoxicity, accompanied by elevated levels of cleaved caspase-3 and PARP1, highlighting its role in driving cell death resistance in CCA. Furthermore, DOCK1 inhibition disrupted EGF-stimulated EGFR phosphorylation and downstream signaling through Erk1/2, a critical pathway supporting tumor growth, survival, and therapeutic resistance. These findings position DOCK1 as a central enhancer of EGFR-mediated oncogenic signaling, playing a key role in the aggressive phenotype and therapeutic resistance of CCA. Targeting DOCK1 not only suppressed tumor cells proliferation but also enhanced cell death, offering a dual therapeutic advantage. Our study provides strong evidence supporting DOCK1 as a promising therapeutic target and suggests that its inhibition, particularly in combination with EGFR-targeted therapies, could overcome EGFR mutant and drug resistance mechanisms and improve clinical outcomes in CCA. By unveiling this novel therapeutic strategy, our research lays the foundation for advancing more effective and stable treatments for this aggressive and often deadly malignancy. Citation Format: Kishor Pant, Kory Engelstad, Estanislao Peixoto, Ainhoa Lapitz, Jesus M. Banales, Adrian Mansini, Sergio A. Gradilone. DOCK1 as a therapeutic target for EGFR resistance in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6808.

Abstract 4387: Identification of new drug combinations that cooperatively target KRAS-dependent nuclear protein export to treat mutant KRAS-driven pancreatic adenocarcinoma

Abstract Metastatic pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor prognosis. Identifying useful drug combinations for PDAC treatment is an important challenge for molecularly targeted therapy. Oncogenic mutations in the KRAS gene, which are found in over 90% of PDAC patients, play a critical role in PDAC. Although KRAS inhibitors may be found useful in PDAC treatment, it is likely that resistance will develop in most patients given single agent treatment. To overcome this anticipated limitation, effective drug combinations that include KRAS inhibitors will be necessary. We have recently determined in lung cancer cells that RAS facilitates the export of nuclear proteins into the cytoplasm via a mechanism independent of canonical RAS signaling pathways (Tripathi et al, Nature Cancer, 2024). This suggests that new drug combinations that cooperate with the inhibition of the RAS nuclear export function might be suitable for cancer therapy. Our preclinical PDAC studies suggest the lung cancer mechanisms are relevant to PDAC, with implications for possible new drug combinations. In PDAC lines, we have found that the DLC1 tumor suppressor protein is a critical downstream target of KRAS-dependent nuclear protein export. Perinuclear binding of KRAS-GTP to RanGAP1 promotes the hydrolysis of RAN-GTP to RAN-GDP and the consequent release of nuclear protein cargo into the cytoplasm. Export of the nuclear EZH2 methyltransferase leads to methylation of the DLC1 protein, making it susceptible to proteasomal degradation. Conversely, KRAS inhibition prevents the nuclear export of EZH2, leading to an increase in DLC1 protein levels. Consistent with these findings, analysis of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database indicates DLC1 protein levels in PDAC are lower than would be expected from the relatively high DLC1 mRNA expression in these tumors. In the adjacent normal tissues, CPTAC indicates EZH2 protein levels are significantly lower and DLC1 protein levels are significantly higher than in the tumors. Our preclinical studies indicate that a three-drug combination consisting of a KRAS inhibitor plus AKT and SRC inhibitors have significantly more antitumor activity than the KRAS inhibitor alone. It is likely that the cooperation of the three-drug combination is attributable to the KRAS inhibitor increasing DLC1 protein levels, while the inhibitors of AKT kinase and SRC kinase reverse and prevent the direct phosphorylation and attenuation of DLC1 tumor suppressor functions by these kinases, thereby reactivating the tumor suppressor activity of DLC1 protein, which enhances the antitumor activity against PDAC with mutant KRAS. In addition to this useful three-drug combination, we are currently testing other drug combinations that take advantage of the KRAS-mediated nuclear protein export function. Citation Format: Brajendra K. Tripathi, Sophia M. Shahin, Xiaolan Qian, Marian E. Durkin, Ross Lake, James H. Doroshow, Dunrui Wang, Douglas R. Lowy. Identification of new drug combinations that cooperatively target KRAS-dependent nuclear protein export to treat mutant KRAS-driven pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4387.

Abstract 3304: HCAR2 activation limits tumor growth by the activation of p53 signaling in breast cancer

Abstract Breast cancer (BC) is the most common cause of cancer-related death in women. BC affects ∼2.3 million women worldwide, with ∼685, 000 deaths every year. Therefore, a clear understanding of the molecular mechanism(s) associated with BC formation and the development of more effective therapeutic strategies for the prevention and treatment of this disease are sorely needed. The p53 tumor suppressor protein, encoded by the TP53 gene, prevents multicellular organisms from developing cancer. However, p53 mutations occur in almost all tumors, emphasizing its role as a formidable barrier that must be breached to allow oncogenic transformation. p53 is the most frequently mutated gene in BC, especially in triple-negative breast cancer (TNBC). Although p53 mutation has been seen in 18-25% of primary BC, about 80% of TNBC is associated with p53 mutation or inactivation. p53 is a multi-functional protein that controls the cell cycle, cell proliferation, DNA repair, senescence, and apoptosis, thereby blocking the proliferation of damaged cells and preventing tumor formation. Therefore, the development of therapeutic strategies that will rejuvenate wild-type p53 (WTp53) function or target mutant p53 (p53α) should have a high clinical impact on the prevention and treatment of BC. We found that HCAR2 (Hydroxycarboxylic acid receptor 2) or NIACR1 (Niacin receptor 1) or HM74A or GPR109A, a G-protein coupled receptor for the vitamin B3 Niacin (NA), the bacterial fermentation product butyrate (BTR), and the ketone body β-hydroxybutyrate (BHB), activates WTp53 and also destabilizes p53α in BC cells. p53 is an NAD+-dependent molecule, and deficiency of NA, the precursor of NAD+ and HCAR2 agonist impairs p53 function. NA- and BTR-induced HCAR2 activation can enhance p53 function by decreasing cAMP, thereby attenuating the binding of MDM2 to p53 and the resulting proteasomal degradation and inhibiting SIRT1, a p53 deacetylase. Similarly, BHB, an HCAR2 agonist and the primary ketone body in mammals, potentiates p53 function by increasing p53 acetylation. Further, HCAR2 activation induces cytotoxicity in p53α BC cells via inhibition of HSP90-HDAC chaperone, which stabilizes the transcription of p53α in the mammary epithelium. Therefore, HCAR2 activation should have a high clinical translational potential for the prevention and treatment of BC by rejuvenating WTp53 and destabilizing p53α in BC cells. Overall, our study provides preclinical evidence to show the functional implication of ketogenic signaling in the regulation of p53 and its relevance to BC prevention and treatment. Citation Format: SONIA BATAN, Nanditi Thangaraju, Jabuneesa Khanom, Kunal Kumar, Snigdha Ganjikunta, Subash Sundaram, Nikhil Patel, Santhakumar Manicassamy, Puttur D. Prasad, Muthusamy Thangaraju. HCAR2 activation limits tumor growth by the activation of p53 signaling in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3304.

Abstract 3162: New hit compounds for p53 identified with a CETSA based high throughput screen

Abstract The p53 tumor suppressor protein is of central importance in tumor biology because of its role as guardian against neoplastic growth. The importance of p53 is highlighted by the frequency of mutations that have been identified in cancers. Most of these mutations are inactivating the original functions of the protein but some mutations also provide p53 with new functions that act pro-tumorigenic. Therefore, therapeutic strategies and initiatives that either restore the original function of p53 or limit gained functions have been applied. Numerous compounds have been reported in the literature to modulate p53 function. Still, to date, no project has proven successful in the clinical setting, which highlights the difficulty of finding compounds to manipulate p53. Reasons for the undruggable nature of p53 comprise the absence of accessible deep pockets and its lack of enzymatic activity. To understand the nature of the interaction between a set of reference compounds and p53, we subjected these molecules to a high throughput (HT) assay based on the Cellular Thermal Shift Assay (CETSA). Three different breast cancer cell lines with different p53 mutational status were used for this study: SK-BR-3 (p53R175H), BT-474 (p53E285K), and MCF-7 (p53WT). We generated ten-point concentration - response curves for all compounds in each of the three cell lines. In addition, knowing the complex nature of both p53 and its central role in the cell, we ran the assay both in lysate and intact cells. Target engagement could be established for some of the compounds in intact cells but was lacking in lysate. There was also a cell type specific variation in effects of the compounds. As an example, Butein exhibited single digit micromolar potency in intact SK-BR-3 cells, whereas there was very little effect in MCF-7 cells, which could be attributed to the difference in p53 mutational status between these cells. To learn more about the reference compounds and to investigate if their effects could be attributed to other cellular protein interactions, we subjected them to a proteome wide CETSA profiling, using mass spectrometry as a read out. The target engagement profile of the tested compounds was as diverse as their chemical structures, with very little overlap. Next, we again turned to the CETSA HT platform to screen a library of low molecular weight compounds aiming to identify new hit compounds for mutant p53. The screen was successful in identifying compounds that induced a thermal shift of mutant p53 in intact SK-BR-3 cells. These hits were then confirmed by QPCR based efficacy assays which showed effects on p53 regulated transcription. Here we highlight the applicability of CETSA in investigating the relevance of annotated tool compounds and how the properties of CETSA make it well-suited for screening for new hit matter, also when aiming for notoriously hard to drug targets. Citation Format: Tomas Friman, Merve Kacal, Laurence Arnold, Stina Lundgren, Daniel Martinez Molina. New hit compounds for p53 identified with a CETSA based high throughput screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3162.

Abstract 7015: Intelligent molecular glue discovery by identifying glueable ligase-target pairs at scale

Abstract Targeted protein degradation can be induced by monovalent small molecules, called molecular glues. These molecules modify the substrate-binding interface of E3 ubiquitin ligases and reroute their binding to novel targets, opening the possibility of therapeutically inhibiting proteins previously considered “undruggable”. However, for a molecular glue to sufficiently stabilize a ligase-target interaction and induce degradation, the two proteins must exhibit proper shape complementarity and contain productive amino acid contacts - two proteins selected at random are unlikely to be glued by any small molecule. Identifying novel “glueable” ligase-target pairs requires the discovery and characterization of weak interactions (likely in the micromolar affinity range) between thousands or more potential pairs of human E3 ligases and therapeutically relevant target proteins. The need to evaluate vast numbers of potential protein interactions while maintaining exquisite sensitivity for weak interactions presents a major technical roadblock. To address this challenge, we apply AlphaSeq, a high-throughput and highly sensitive experimental platform for measuring protein-protein interactions, to discover and structurally characterize weak interactions between ligases and target proteins. We validate our approach by characterizing known ligase-target interactions, such as between the E3 ligase MDM2 and the tumor suppressor protein p53. We then leverage AlphaSeq for the discovery of molecular glues for novel ligase-target pairs and orthogonally validate these interactions with surface plasmon resonance (SPR). Finally, we use AlphaSeq mutational data to structurally characterize the interaction interface and inform the intelligent discovery of molecular glues. Citation Format: Phil Burke, Arpita Sen, David Noble, Abhishek Dogra, Ruchi Bansal, Dasha Krayushkina, Natasha Seelam Murakowska, Kerry McGowan, Charles Lin, Yeweon Lee, Randolph Lopez. Intelligent molecular glue discovery by identifying glueable ligase-target pairs at scale [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7015.

Abstract 1394: Apoptosis of pancreatic cancer cells after co-treatment with eugenol and tumor necrosis factor-related apoptosis-inducing ligand

Abstract Pancreatic cancer is a refractory cancer with limited treatment options, and resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) poses a significant therapeutic challenge. This study aimed to investigate whether eugenol, the main component of clove oil with known anticancer, anti-inflammatory, and antioxidant properties, could enhance TRAIL sensitivity by upregulating death receptor (DR) expression levels in pancreatic cancer cells. To explore this, we treated pancreatic cancer cells with eugenol and TRAIL, individually and in combination, and evaluated apoptosis and cell proliferation rates. Expression levels of key proteins, including DR5, FLICE-inhibitory protein (FLIP), and p53, were assessed. Additionally, reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, and C/EBP-homologous protein (CHOP) involvement were analyzed using siRNA knockdown approaches. Our findings revealed that combined treatment with eugenol and TRAIL significantly increased apoptosis and inhibited cell proliferation compared with eugenol alone. Eugenol upregulated DR5 expression, downregulated the anti-apoptotic protein FLIP, and increased levels of the tumor suppressor protein p53. Furthermore, eugenol induced ROS generation and caused ER stress, with CHOP playing a critical role. CHOP knockdown decreased DR5 expression and reduced the synergistic apoptotic effects of eugenol and TRAIL, confirming the ROS-mediated ER stress-CHOP pathway as a key mechanism. These results demonstrate that eugenol enhances TRAIL-induced apoptosis by upregulating DR5 through ROS-mediated ER stress and CHOP activation, which further enhances ER stress via p53 induction and FLIP downregulation. This suggests that eugenol has the potential to serve as a novel TRAIL sensitizer and offers a promising approach to overcoming TRAIL resistance in pancreatic cancer treatment. Citation Format: Hyun Hee Kim, Dae-Hee Lee. Apoptosis of pancreatic cancer cells after co-treatment with eugenol and tumor necrosis factor-related apoptosis-inducing ligand [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1394.

Abstract 1930: Advancing cervical cancer early detection: A novel lateral flow immunoassay targeting p16INK4a

Cervical cancer ranks as the fourth most prevalent cancer among women globally, with a stark disparity in disease burden and mortality between high-income and low- to middle-income countries (LMICs). Sub-Saharan Africa faces the highest incidence and mortality rates, primarily due to limited access to preventive measures such as vaccines, screening, early detection, and treatment. With approximately 90% of cervical cancer cases occurring in LMICs, there is an urgent need for effective, affordable screening solutions. This study addresses this critical gap by developing a cost-effective, easy-to-use point-of-care (POC) screening test. Specifically, we aim to create a lateral flow immunoassay (LFIA) to detect the overexpression of cyclin-dependent kinase inhibitor 2A (CDKN2A or p16INK4a), a key tumor suppressor protein overexpressed in cervical cancer. The LFIA is designed to be capable of detecting p16INK4a in precancerous and cancerous cells from cervical swab samples, thereby providing an accessible and efficient tool for cervical cancer screening in resource-limited settings. The developed POC LFIA utilizes antigen-antibody interactions. It has four key components: a sample pad, a conjugate pad, a reaction membrane, and an absorbent pad. Cervical swab samples applied to the sample pad rehydrate the dried conjugates, allowing them to interact with the analyte and migrate to the reaction zone, where a gold nanoparticle (AuNP)-conjugated antibody facilitates detection. Initial validation of p16INK4a levels in clinical swab samples was performed using an enzyme-linked immunosorbent assay, which revealed significant overexpression of p16INK4a in high-grade squamous intraepithelial lesions (HSIL), confirming its role as a biomarker for cervical precancerous conditions. Biolayer interferometry was used to select antibodies with optimal nanomolar to picomolar binding affinities. The chosen antibody was conjugated with AuNPs through passive adsorption, achieving a stable 100:1 molar antibody-to-AuNP ratio over seven days. The test and control lines were optimized using in-house dipsticks, with 1.5 mg/mL anti-mouse immunoglobulin G proving ideal for the control line. The current test can reliably detect p16INK4a concentrations as low as 0.5 μg/mL, with further refinements underway to lower the detection limit for clinical use. This simple and cost-effective LFIA offers robust performance, addressing the critical need for affordable and accessible cervical cancer screening, particularly in LMICs. With continued optimization, this tool holds significant promise for improving early detection and treatment outcomes in resource-limited settings. Keywords: point-of-care test, cervical cancer, p16INK4a, early detection, prevention, LMICs Citation Format: Samrin F. Habbani,Sayeh J. Dowlatshahi,Scott C. Bolton,Lucy T. Tecle,Lisa Flowers,Jacqueline C. Linnes,Sulma I. Mohammed. Advancing cervical cancer early detection: A novel lateral flow immunoassay targeting p16INK4a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1930.

Abstract 1413: Dissecting the biology of mutant p53 with the Cellular Thermal Shift Assay (CETSA)

The tumor suppressor protein p53 is one of the most important safeguards against malignant transformation of mammalian cells, but it is also one of the most frequently mutated proteins in cancers. Therefore, p53 constitutes an attractive drug target in oncology. However, there has been little success in finding clinical candidate drugs that directly manipulate the function of p53. The p53 protein offers several challenges for investigators wanting to manipulate its function e.g., the absence of accessible binding pockets, lack of enzymatic activity, and its intracellular location. Moreover, another complication with p53 is the plethora of mutations that alter the protein’s characteristics. Most mutational changes in p53 result in a loss of function, but there are also examples of gain of function mutations in p53. We have applied the Cellular Thermal Shift Assay (CETSA) for screening of new chemical hit matter for binding to p53. As CETSA exploits the compound induced changes in protein melting behavior it is important to know the melting characteristics of the protein under investigation. Since p53 displays a wide variety of mutational variants, we decided to investigate three different variants present in three separate breast cancer cell lines: SK-BR-3 (p53R175H), BT-474 (p53E285K), and MCF-7 (p53WT). The melting behavior of p53 protein was similar irrespective of genomic status of p53 in intact cells. However, in lysed cells there was striking difference in p53 protein melting between MCF-7 cells that carry WT p53 and the two other breast cancer cell lines that carry mutations in the gene coding p53. The melting temperature for WT p53 was nearly the same in both lysate and intact cells, whereas there was 3 to 4°C difference between intact cells and lysates derived from the two cell lines that expressed mutated p53. Both of the mutated forms of p53 found in SK-BR-3 cells and BT-474 cells displayed greater thermal stability in lysate compared to MCF-7. Intrigued by this difference we set out to investigate its cause. It is known that certain mutated forms of p53 are rescued from proteasomal degradation by associating with chaperone proteins like Hsp90. Therefore, the different breast cancer cell lines were treated with inhibitors of Hsp90 function and protein melting of the respective form of p53 was assessed. Hsp90 modulation by Geldanamycin had a much more potent effect on thermal stability of mutant p53 compared to the WT counterpart, which indicates that Hsp90 could be involved in conferring increased thermal stability of mutant p53. Citation Format: Tomas Friman, Merve Kacal, Laurence Arnold, Daniel Martinez Molina. Dissecting the biology of mutant p53 with the Cellular Thermal Shift Assay (CETSA) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1413.

Abstract 3521: A novel T cell engager targeting HLA-A02:01 TP53-R175H for cancer immunotherapy

Background: The tumor suppressor protein p53 (TP53) plays a pivotal role in preventing tumor formation by inducing cell cycle arrest and apoptosis in response to DNA damage. However, TP53 mutations are prevalent across various cancer types, and these mutations not only result in loss of tumor suppressive functions but also confer gain-of-function properties that drive oncogenesis. Among these mutations, HLA-A0201 TP53-R175H represents the largest TP53 population with >34,000 cases per year in the US and EU across all solid tumor indications, with no therapies explicitly targeting this mutation in the clinic. Leveraging Affini-T’s TETHER™ platform, we present the development of a novel TCR-based T cell engager designed to redirect the cytotoxic potential of T cells against mutant TP53-expressing cancer cells. Methods: A bispecific T cell engager was engineered to simultaneously engage T cells via CD3 and target TP53-R175H-expressing cancer cells by an affinity-matured specific TCR. Affinity maturation of the TCR was performed to increase its specificity and binding affinity towards the mutant peptide. Tumor co-culture assays were conducted to evaluate T cell-mediated cytotoxicity. T cell activation was assessed by measuring cytokine production. The tolerability profile of the engagers was established by X-scan, evaluating T cell activation by normal tissue. Results: The data demonstrate successful affinity maturation of the TCR, resulting in enhanced recognition of the HLA-A0201 TP53-R175H peptide with high specificity. Tumor co-culture assays revealed potent T cell-mediated killing of mutant TP53-R175H-expressing cancer cells by the engager, accompanied by robust T cell activation characterized by cytokine production. Importantly, the engager exhibited favorable tolerability profiles, demonstrated by minimal off-target activation via X-scan and with minimal T cell activation towards normal tissue. Conclusions: These findings highlight the promising activity and tolerability profile of a novel T cell engager targeting HLA-A02 TP53-R175H for cancer immunotherapy. Harnessing the cytotoxic potential of T cells against mutant TP53-expressing tumors presents a promising approach for the development of innovative cancer treatments. Citation Format: Mark Ng, Emily Zygiel, Alexander McQuown, Jinsheng Liang, Luhua Shen, Joshua Francis, Xingyue He, Hubert Lam, Gary Shapiro, Loic Vincent. A novel T cell engager targeting HLA-A02:01 TP53-R175H for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2521.

Abstract 1776: Investigation into the enhancement of 20S proteasome-mediated proteolysis as a therapeutic for HPV-associated cervical cancer

Abstract Human papillomaviruses (HPVs) are one of the most common sexually transmitted infections (STIs) in the world. HPVs can be divided into two main categories, low-risk and high-risk. Low-risk HPVs do not cause cancer and can often be cleared by the infected person’s immune system. However, high-risk HPVs are responsible for approximately 5% of cancers worldwide - most commonly cervical cancer, and there is currently no cure once infection occurs. While prophylactic vaccines exist that can successfully prevent the infection of many high-risk HPVs, vaccination rates worldwide are low. After infection, the virus incorporates its genome into the host cell genome, and the cell begins to produce HPV proteins. In high-risk HPVs, two of these proteins, HPV E6 and E7, are oncoproteins with partially disordered structures. E6 and E7 proteins have many functions impacting cancer development, including inducing the degradation of tumor suppressor proteins p52 and pRB, respectively, through the ubiquitin-26S proteasome system (UPS). The removal of these cell cycle progression checkpoints allows for uncontrolled cell growth, which can promote cancer. The Tepe lab works on the development of small molecules which can increase the protein degradation capacity of the 20S proteasome, a protease that specifically degrades disordered proteins in a ubiquitin-independent manner. This project works to establish if the 20S proteasome can degrade HPV E6 and E7 proteins, and if the use of 20S proteasome enhancing small molecules can deplete levels in HPV+ cervical cancer cells. We have discovered that treatment with two of these compounds decreases E6 and E7 levels in a HPV18+ cervical cancer cell line and work is ongoing to determine if this impact is through 20S proteasome activation. Additionally, we are investigating if this decrease is enough to inhibit the out-of-control cell growth caused by E6 and E7. The goal of this project is to understand if 20S proteasome activators can be used as a therapy for HPV-mediated cervical cancer. Citation Format: Shannon M. Cartwright, Linh Le, Kate Hartnett, Jetze J. Tepe. Investigation into the enhancement of 20S proteasome-mediated proteolysis as a therapeutic for HPV-associated cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1776.