P53 Function Research Articles

Decreased expression of p53 is associated with down expression of zyxin in breast cancer.

Breast cancer (BC) is considered one of the most common malignant tumors leading to death in women, and genetic factors have a crucial role in BC pathogenesis. Zyxin (ZYX) is one of these factors that may be important in p53 level and function. Thus, the present work aimed to investigate the ZYX gene and protein expression in tumor tissue and matched margin tissue and its correlation with the p53 expression. In a present case-control study, 30 tumors and 30 matched margin tissues were obtained from Iran Tumor Bank/Tehran University of Medical Sciences. Real-time polymerase chain reaction and western blot analysis techniques were applied to evaluate the genes and protein expression, respectively. The data showed that expression of the ZYX gene in tumor tissues significantly decreased (p = 0.0274) compared to matched margin tissues. In contrast, the p53 gene expression in tumor tissues had no significant difference with matched margin tissues. Additionally, we observed that ZYX and p53 genes expression in tumor tissues of estrogen receptor-positive patients had significant elevation than estrogen receptor-negative patients (p < 0.001, p < 0.001, respectively). The data of the western blot analysis technique showed that protein expression of ZYX (p = 0.0024) and P53 protein (p = 0.0218) in tumor tissues was significantly reduced compared to matched margin tissues. Additionally, our analysis showed a direct and significant correlation between the expression of ZYX and p53 proteins (r = 0.7797, p = 0.0126) and expression of ZYX and p53 genes (r = 0.3079, p = 0.0187). Based on our observation, ZYX might have a tumor suppressor role and is associated with p53.

Activating transcription factor 3 mediates apoptosis and cell cycle arrest in TP53-mutated anaplastic thyroid cancer cells

BackgroundIt is believed that loss of p53 function plays a crucial role in the progression of well to poorly differentiated thyroid cancers including anaplastic thyroid carcinoma (ATC). Given the poor prognosis of ATC due to its strong therapeutic resistance, there is a need to establish new therapeutic targets to extend the survival of ATC patients. Activating transcription factor 3 (ATF3) can inhibit the oncogenic activity of mutant p53 and, as a result, contribute to tumor suppression in several TP53-mutated cancers. Herein, we demonstrate that the ectopic overexpression of ATF3 leads to the suppression of oncogenic mutant p53 activity in chemo-resistant 8305 C thyroid cancer cells harboring R273C p53 gene mutation.MethodsThe biological behavior of 8305 C cells was assessed pre- and post-transfection with pCMV6-ATF3 plasmid using MTT assay, fluorescent microscopy, cell cycle, and annexin V/PI flow cytometric analysis. The effect of ectopic ATF3 overexpression on the cellular level of p53 was examined by western blotting assay. The mRNA expression levels of TP53, TAp63, ΔNp63, and SHARP1 were evaluated in ectopic ATF3-expressing cells compared to controls.ResultsThe overexpression of ATF3 in 8305 C thyroid cancer cells significantly decreased cell viability and induced apoptosis and cell cycle arrest in vitro. The immunoblotting of p53 protein revealed that ATF3 overexpression significantly increased the level of mutant p53 in 8305C cells compared to mock-transfected control cells. Additionally, elevated mRNA levels of TAp63 and SHARP1 and a decreased mRNA level of ΔNp63 were observed in PCMV6-AC-ATF3-transfected 8305 C cells with significant differences compared to the mock and untreated cells.ConclusionIn light of our findings, it is evident that therapeutic strategies aimed at increasing ATF3 expression or enhancing the interaction between ATF3 and mutant p53 can be a promising approach for the treatment of p53-mutated metastatic thyroid cancer.

MDM2 inhibitor APG-115 synergizes with ABT-199 to induce cell apoptosis in chronic lymphocytic leukemia.

Although clinical outcomes in chronic lymphocytic leukemia (CLL) have greatly improved with several approved small molecular inhibitors, acquired resistance does occur, leading to disease progression and eventual death. Thus, the effort to explore novel inhibitors and combination therapeutic regimens is needed. The inhibition of MDM2-p53 interaction to restore p53 function has been regarded as a potential strategy for treating different cancers. We investigated the effects of novel MDM2 inhibitor APG-115 in CLL. We found that APG-115 treatment upregulated the expression of p53, MDM2, and p21 at the mRNA and protein level. APG-115 inhibited cell proliferation, induced apoptosis, and arrested the cell cycle at G0/G1 stage. Moreover, APG-115 inhibited the expression of BCL-2, BCL-xL, and MCL-1, and suppressed the activation of AKT and ERK signaling pathways. APG-115 combined with the BCL2 inhibitor, ABT-199 (venetoclax), led to further inhibition of the expression of BCL-2 family anti-apoptotic proteins and consequently enhanced cell death. Collectively, this study demonstrates that APG-115 activates p53 and thus inhibits multiple pro-survival mechanisms, which provides a rational explanation for APG-115 efficiency in inducing cell apoptosis in CLL. The synergistic effect of APG-115 with ABT-199 suggested a potential combination application in CLL therapy.

Functional evaluation of germline TP53 variants identified in Brazilian families at-risk for Li–Fraumeni syndrome

Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li–Fraumeni (LFS). Variants affecting its activity can drive tumorigenesis altering p53 pathways and their identification is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G > C, c.320A > G, c.417G > T, c.460G > A, c,522G > T, c.589G > A and c.997C > T) identified in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed significantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited effect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classification as pathogenic variants. This underscores the significance of conducting functional studies on germline TP53 missense variants classified as variants of uncertain significance to ensure proper management of LFS-related cancer risks.

Unraveling the Role of TP53 in Colorectal Cancer Therapy: From Wild-Type Regulation to Mutant.

The p53, a pivotal tumor suppressor, regulates various cellular responses, including DNA repair and apoptosis. Normally, p53 levels are low due to murine double minute clone 2 (MDM2) mediated polyubiquitination. However, stress signals disrupt p53-MDM2 interaction, stabilizing p53 and activating target genes. Dysfunctional p53 is common in cancers, especially colorectal cancer (CRC), with TP53 mutations in 43% of tumors. These mutations impair wild-type p53 function or confer novel activities, promoting cancer progression. Despite drugs targeting p53 entering trials, understanding wild-type and mutant p53 functions is crucial for novel CRC therapies. P53 mutations not only impact DNA repair and apoptosis but also play a crucial role in tumor immunotherapy. While rendering tumors resistant to chemotherapy, p53 mutations provide opportunities for immunotherapy due to neoantigen-rich tumors. Additionally, p53 mutations influence tumor microenvironment cells, such as fibroblasts and immunosuppressive cells, through p53-mediated signaling pathways. Investigating p53 mutations in tumor therapy is vital for personalized medicine and immunotherapy. In cancer treatment research, scientists explore drugs and strategies to restore or enhance p53 function. Targeting wild-type p53 aims to restore DNA repair and cell cycle control, while targeting mutant p53 seeks new drugs to inhibit its detrimental effects, advancing tumor treatment. Understanding p53 drugs and strategies is crucial for cancer therapy progress.

Ribosomal Protein S4 X-Linked as a Novel Modulator of MDM2 Stability by Suppressing MDM2 Auto-Ubiquitination and SCF Complex-Mediated Ubiquitination.

Mouse double minute 2 (MDM2) is an oncoprotein that is frequently overexpressed in tumors and enhances cellular transformation. Owing to the important role of MDM2 in modulating p53 function, it is crucial to understand the mechanism underlying the regulation of MDM2 levels. We identified ribosomal protein S4X-linked (RPS4X) as a novel binding partner of MDM2 and showed that RPS4X promotes MDM2 stability. RPS4X suppressed polyubiquitination of MDM2 by suppressing homodimer formation and preventing auto-ubiquitination. Moreover, RPS4X inhibited the interaction between MDM2 and Cullin1, a scaffold protein of the Skp1-Cullin1-F-box protein (SCF) complex and an E3 ubiquitin ligase for MDM2. RPS4X expression in cells enhanced the steady-state level of MDM2 protein. RPS4X was associated not only with MDM2 but also with Cullin1 and then blocked the MDM2/Cullin1 interaction. This is the first report of an interaction between ribosomal proteins (RPs) and Cullin1. Our results contribute to the elucidation of the MDM2 stabilization mechanism in cancer cells, expanding our understanding of the new functions of RPs.

Skeletal muscle p53-depletion uncovers a mechanism of fuel usage suppression that enables efficient energy conservation.

The ability of skeletal muscle to respond adequately to changes in nutrient availability, known as metabolic flexibility, is essential for the maintenance of metabolic health and loss of flexibility contributes to the development of diabetes and obesity. The tumour suppressor protein, p53, has been linked to the control of energy metabolism. We assessed its role in the acute control of nutrient allocation in skeletal muscle in the context of limited nutrient availability. A mouse model with inducible deletion of the p53-encoding gene, Trp53, in skeletal muscle was generated using the Cre-loxP-system. A detailed analysis of nutrient metabolism in mice with control and knockout genotypes was performed under ad libitum fed and fasting conditions and in exercised mice. Acute deletion of p53 in myofibres of mice activated catabolic nutrient usage pathways even under ad libitum fed conditions, resulting in significantly increased overall energy expenditure (+10.6%; P=0.0385) and a severe nutrient deficit in muscle characterized by depleted intramuscular glucose and glycogen levels (-62,0%; P<0.0001 and -52.7%; P<0.0001, respectively). This was accompanied by changes in marker gene expression patterns of circadian rhythmicity and hyperactivity (+57.4%; P=0.0068). These metabolic changes occurred acutely, within 2-3days after deletion of Trp53 was initiated, suggesting a rapid adaptive response to loss of p53, which resulted in a transient increase in lactate release to the circulation (+46.6%; P=0.0115) from non-exercised muscle as a result of elevated carbohydrate mobilization. Conversely, an impairment of proteostasis and amino acid metabolism was observed in knockout mice during fasting. During endurance exercise testing, mice with acute, muscle-specific Trp53 inactivation displayed an early exhaustion phenotype with a premature shift in fuel usage and reductions in multiple performance parameters, including a significantly reduced running time and distance (-13.8%; P=0.049 and -22.2%; P=0.0384, respectively). These findings suggest that efficient nutrient conservation is a key element of normal metabolic homeostasis that is sustained by p53. The homeostatic state in metabolic tissues is actively maintained to coordinate efficient energy conservation and metabolic flexibility towards nutrient stress. The acute deletion of Trp53 unlocks mechanisms that suppress the activity of nutrient catabolic pathways, causing substantial loss of intramuscular energy stores, which contributes to a fasting-like state in muscle tissue. Altogether, these findings uncover a novel function of p53 in the short-term regulation of nutrient metabolism in skeletal muscle and show that p53 serves to maintain metabolic homeostasis and efficient energy conservation.

The anti-tumor effect of trifluridine via induction of aberrant mitosis is unaffected by mutations modulating p53 activity

The fluorinated thymidine analog trifluridine (FTD) is a chemotherapeutic drug commonly used to treat cancer; however, the mechanism by which FTD induces cytotoxicity is not fully understood. In addition, the effect of gain-of-function (GOF) missense mutations of the TP53 gene (encoding p53), which promote cancer progression and chemotherapeutic drug resistance, on the chemotherapeutic efficacy of FTD is unclear. Here, we revealed the mechanisms by which FTD-induced aberrant mitosis and contributed to cytotoxicity in both p53-null and p53-GOF missense mutant cells. In p53-null mutant cells, FTD-induced DNA double-stranded breaks, single-stranded DNA accumulation, and the associated DNA damage responses during the G2 phase. Nevertheless, FTD-induced DNA damage and the related responses were not sufficient to trigger strict G2/M checkpoint arrest. Thus, these features were carried over into mitosis, resulting in chromosome breaks and bridges, and subsequent cytokinesis failure. Improper mitotic exit eventually led to cell apoptosis, caused by the accumulation of extensive DNA damage and the presence of micronuclei encapsulated in the disrupted nuclear envelope. Upon FTD treatment, the behavior of the p53-GOF-missense mutant, isogenic cell lines, generated by CRISPR/Cas9 genome editing, was similar to that of p53-null mutant cells. Thus, our data suggest that FTD treatment overrode the effect on gene expression induced by p53-GOF mutants and exerted its anti-tumor activity in a manner that was independent of the p53 function.

Microarray Analysis of Tumor Suppressor Proteins p53 and p63: Their Role in Tumor Cell Dynamics

Background: Microarray technology has become essential in cancer research, providing insights into gene expression patterns and regulatory networks governed by key transcription factors. This review focuses on using microarray technology to elucidate genes regulated by tumor suppressor proteins p53 and p63. These proteins maintain genomic stability, regulate the cell cycle, and induce programmed cell death. Dysregulation of p53 and p63 is common in various cancers, highlighting their significance in cancer biology. Methods: Microarray technology enables high-throughput analysis of thousands of genes simultaneously. Researchers compare gene expression profiles in cancer cells and tissues with functional p53 and p63 against those with impaired function. This comparative analysis identifies numerous downstream target genes and pathways influenced by these transcription factors. Results: Microarray studies have revealed a wide array of genes regulated by p53 and p63, involved in DNA repair, apoptosis, cell cycle control, and epithelial differentiation. These findings enhance our understanding of molecular mechanisms driving cancer initiation, progression, and therapeutic resistance. Additionally, microarray technology enables the stratification of cancer subtypes based on distinct gene expression signatures associated with p53 and p63 status, offering insights into patient prognosis, treatment responses, and personalized therapeutic strategies. Conclusion: Microarray technology has significantly advanced our knowledge of gene regulatory networks orchestrated by p53 and p63 in cancer. Despite limitations like data interpretation and cross-hybridization, advancements in bioinformatics and complementary technologies are improving study accuracy and reliability, promising better cancer diagnostics, prognostics, and targeted therapies.

An 8-aminoquinoline-naphthyl copper complex causes apoptotic cell death by modulating the expression of apoptotic regulatory proteins in breast cancer cells

Breast cancer is one of the most common cancers globally and a leading cause of cancer-related deaths among women. Despite the combination of chemotherapy with targeted therapy, including monoclonal antibodies and kinase inhibitors, drug resistance and treatment failure remain a common occurrence. Copper, complexed to various organic ligands, has gained attention as potential chemotherapeutic agents due to its perceived decreased toxicity to normal cells. The cytotoxic efficacy and the mechanism of cell death of an 8-aminoquinoline-naphthyl copper complex (Cu8AqN) in MCF-7 and MDA-MB-231 breast cancer cell lines was investigated. The complex inhibited the growth of MCF-7 and MDA-MB-231 cells with IC50 values of 2.54 ± 0.69 μM and 3.31 ± 0.06 μM, respectively. Nuclear fragmentation, annexin V binding, and increased caspase-3/7 activity indicated apoptotic cell death. The loss of mitochondrial membrane potential, an increase in caspase-9 activity, the absence of active caspase-8 and a decrease of tumour necrosis factor receptor 1(TNFR1) expression supported activation of the intrinsic apoptotic pathway. Increased ROS formation and increased expression of haem oxygenase-1 (HMOX-1) indicated activation of cellular stress pathways. Expression of p21 protein in the nuclei was increased indicating cell cycle arrest, whilst the expression of inhibitor of apoptosis proteins (IAPs); cIAP1, XIAP and survivin were decreased, creating a pro-apoptotic environment. Phosphorylated p53 species; phospho-p53(S15), phospho-p53(S46), and phospho-p53(S392) accumulated in MCF-7 cells indicating the potential of Cu8AqN to restore p53 function in the cells. In combination, the data indicates that Cu8AqN is a useful lead molecule worthy of further exploration as a potential anti-cancer drug.

Mixtures of Three Mortaparibs with Enhanced Anticancer, Anti-Migration, and Antistress Activities: Molecular Characterization in p53-Null Cancer Cells.

Mortalin, a member of the Hsp70 family of proteins, is commonly enriched in many types of cancers. It promotes carcinogenesis and metastasis in multiple ways of which the inactivation of the tumor suppressor activity of p53 has been firmly established. The downregulation of mortalin and/or disruption of mortalin-p53 interactions by small molecules has earlier been shown to activate p53 function yielding growth arrest/apoptosis in cancer cells. Mortaparibs (Mortaparib, MortaparibPlus, and MortaparibMild) are chemical inhibitors of mortalin isolated by cell-based two-way screening involving (i) a shift in the mortalin staining pattern from perinuclear (characteristics of cancer cells) to pancytoplasmic (characteristics of normal cells) and (ii) the nuclear enrichment of p53. They have similar structures and also cause the inhibition of PARP1 and hence were named Mortaparibs. In the present study, we report the anticancer and anti-metastasis activity of MortaparibMild (4-[(4-amino-5-thiophen-2-yl-1,2,4-triazol-3-yl)sulfanylmethyl]-N-(4-methoxyphenyl)-1,3-thiazol-2-amine) in p53-null cells. By extensive molecular analyses of cell proliferation, growth arrest, and apoptosis pathways, we demonstrate that although it causes relatively weaker cytotoxicity compared to Mortaparib and MortaparibPlus, its lower concentrations were equally potent to inhibit cell migration. We developed combinations (called MortaparibMix-AP, MortaparibMix-AM, and MortaparibMix-AS) consisting of different ratios of three Mortaparibs for specifically enhancing their anti-proliferation, anti-migration, and antistress activities, respectively. Based on the molecular analyses of control and treated cells, we suggest that the three Mortaparibs and their mixtures may be considered for further laboratory and clinical studies validating their use for the treatment of cancer as well as prevention of its relapse and metastasis.

Structural basis of p53 inactivation by cavity-creating cancer mutations and its implications for the development of mutant p53 reactivators

SummaryThe cavity-creating p53 cancer mutation Y220C is an ideal paradigm for developing small-molecule drugs based on protein stabilization. Here, we have systematically analyzed the structural and stability effects of all oncogenic Tyr-to-Cys mutations (Y126C, Y163C, Y205C, Y220C, Y234C, and Y236C) in the p53 DNA-binding domain (DBD). They were all highly destabilizing, drastically lowering the melting temperature of the protein by 8–17 °C. In contrast, two non-cancerous mutations, Y103C and Y107C, had only a moderate effect on protein stability. Differential stabilization of the mutants upon treatment with the anticancer agent arsenic trioxide and stibogluconate revealed an interesting proximity effect. Crystallographic studies complemented by MD simulations showed that two of the mutations, Y234C and Y236C, create internal cavities of different size and shape, whereas the others induce unique surface lesions. The mutation-induced pockets in the Y126C and Y205C mutant were, however, relatively small compared with that of the already druggable Y220C mutant. Intriguingly, our structural studies suggest a pronounced plasticity of the mutation-induced pocket in the frequently occurring Y163C mutant, which may be exploited for the development of small-molecule stabilizers. We point out general principles for reactivating thermolabile cancer mutants and highlight special cases where mutant-specific drugs are needed for the pharmacological rescue of p53 function in tumors.

A phase (Ph) 0/Ia study of brigimadlin concentration in brain tissue and a non-randomized, open-label, dose escalation study of brigimadlin in combination with radiotherapy (RT) in patients (pts) with newly diagnosed glioblastoma (GBM).

2017 Background: MDM2 inhibits tumor suppressor p53. Brigimadlin, a potent MDM2–p53 antagonist, restores wild-type (wt) p53 function and has shown early efficacy in pts with solid tumors (LoRusso et al Cancer Disc 2023). GBM is an area of unmet need with 5-year survival &lt;10%. In p53 wt GBM pt-derived xenograft models, brigimadlin promotes tumor cell apoptosis and extends survival in combination with RT. Methods: NCT05376800 is a Ph 0/Ia open-label, single-arm trial that aims to measure brigimadlin concentration in brain tumor tissue in pts with histologically or radiologically newly diagnosed GBM eligible for resection (Ph 0) and determine the maximum tolerated dose of brigimadlin plus RT in pts with TP53wt, IDH wt, MGMT promoter unmethylated GBM (Ph Ia). In Ph 0, pts received one brigimadlin dose (30 mg or 45 mg) ~12–24 h before resection. Ph 0 primary endpoints are the measured total concentration and the calculated unbound concentration of brigimadlin in brain tissue homogenate from non-contrast enhancing (NCE) and contrast enhancing (CE) regions. The predefined threshold for trial continuation is 0.5 nmol/L (corresponding to IC50 in GBM cell lines) unbound brigimadlin in CE samples in ≥50% of pts. Brigimadlin concentration was measured using LC/MS and corrected for amount of brigimadlin in residual blood. Unbound concentration was calculated using an in vitro estimate of unbound fraction (fu): 0.654% (rat brain slice). fu in human plasma was 0.22%. Kp,uu (ratio of unbound concentration in brain vs plasma) was calculated. Biomarker testing was performed. Results: Data are available for 11 pts (brigimadlin 30 mg: n=6; 45 mg: n=5). In the 30 mg group, median total brigimadlin concentration in NCE samples was 267 nmol/L (4 samples, range 86–316 nmol/L) and 332 nmol/L (6 samples, range 272–952 nmol/L) in CE samples. In the 45 mg group, median total brigimadlin concentration in NCE samples was 197 nmol/L (5 samples, range 140–347 nmol/L) and 603 nmol/L (5 samples, range 441–905 nmol/L) in CE samples. Unbound concentration exceeded the 0.5 nmol/L threshold in most cases (Table). Post-brigimadlin, an increase in selected p53 target gene expression was observed in CE vs NCE tissue. Conclusions: Unbound brigimadlin concentrations in CE regions in all pts receiving the low dose of 30 mg brigimadlin exceeded the 0.5 nmol/L threshold. Kp,uu in most patients was close to 1 in CE regions. Biomarker data support target engagement in brain tissue. Our findings support continued investigation of brigimadlin in GBM. Recruitment is ongoing. Updated data will be presented. Clinical trial information: NCT05376800 . [Table: see text]

Treatment of patients with dedifferentiated liposarcoma (DDLPS) with the MDM2–p53 antagonist brigimadlin and p53 function: A longitudinal analysis of circulating microRNAs (miRNAs) in a first-in-human phase Ia/Ib study.

Treatment of patients with dedifferentiated liposarcoma (DDLPS) with the MDM2–p53 antagonist brigimadlin and p53 function: A longitudinal analysis of circulating microRNAs (miRNAs) in a first-in-human phase Ia/Ib study.

TP53 Mutations in Acute Leukemias and Myelodysplastic Syndromes: Insights and Treatment Updates.

TP53 mutations are found in 5%-10% of de novo myelodysplastic syndrome (MDS) and AML cases. By contrast, in therapy related MDS and AML, mutations in TP53 are found in up to 30%-40% of patients. The majority of inactivating mutations observed in MDS and AML are missense mutations localized in a few prevalent hotspots. TP53 missense mutations together with truncating mutations or chromosomal loss of TP53 determine a loss-of-function effect on normal p53 function. Clonal expansion of TP53-mutant clones is observed under the selection pressure of chemotherapy or MDM2 inhibitor therapy. TP53-mutant clones are resistant to current chemotherapy, and when responses to treatment have been observed, they have correlated poorly with overall survival. The most heavily investigated and targeted agent for patients with TP53-mutant MDS and AML has been APR-246 (eprenetapopt) a p53 reactivator, in combination with azacitidine, but also in triplets with venetoclax. Despite positive results in phase II trials, a phase III trial did not confirm superior response or improved survival. Other agents, like magrolimab (anti-CD47 antibody), failed to demonstrate improved activity in TP53-mutant MDS and AML. Agents whose activity is not dependent on a functional apoptosis system like anti-CD123 antibodies or cellular therapies are in development and may hold promises. Delivering prognostic information in a dismal disease like TP53-mutated MDS and AML is particularly challenging. The physician should balance hope and realism, describing the trajectory of possible treatments and at the same time indicating the poor outcome, together with promoting adaptive coping in patients and elaborating on the nature of the disease.

Preclinical characterization of pretargeted radioimmunotherapy (PRIT) with GD2-SADA, a self-assembling and disassembling bispecific fusion protein.

e15101 Background: GD2-SADA is a bispecific fusion protein with binding domains for the tumor-associated antigen GD2 and Lutetium 177 (Lu-177)-DOTA complex, and a p53-derived domain that mediates tetramer self-assembly and disassembly (SADA). In Step 1 of PRIT, SADA tetramers bind to GD2+ tumor cells, while unbound protein disassembles into renally cleared monomers. In Step 2, the radioactive payload Lu-177-DOTA is delivered and binds to tumor-bound GD2-SADA. Here, we characterize the binding activities of GD2-SADA to a range of GD2+ tumors and Lanthanide (Ln) metal-DOTA complexes and report the dose responsive, anti-tumor efficacy of GD2-SADA with Lu-177-DOTA in mice. Methods: GD2-SADA binding to GD2+ cell lines (small-cell lung cancer [SCLC], melanoma, osteosarcoma) was evaluated by flow cytometry (FC). A colorimetric competition ELISA was used to investigate binding to Ln-DOTA complexes (terbium [Tb], europium [Eu], and lanthanum [La]), trace metal TM-DOTA complexes (iron [Fe], copper [Cu], or zinc [Zn]), and empty DOTA (used in excess during radiolabeling to ensure complete Lu-177 chelation). GD2 binding of tetrameric GD2-SADA and monomeric p53 mutant SADA was compared using ELISA and FC assays. Tumor response to GD2-SADA with Lu-177-DOTA was evaluated in mice with SC SCLC xenografts (NCI-H524). Mice received 3 weekly treatments with a fixed GD2-SADA dose (10 mg/kg) followed by a Lu-177-DOTA dose (32 mCi or 47mCi/kg; n=6-10 per group). Tumor volumes were monitored weekly for ≥60 days after treatment. Results: GD2+ cell lines showed dose-dependent binding of GD2-SADA, with half-maximal effective concentration (EC50) values between 1.6 and 5 μg/mL. In ELISA and FC assays, the monomeric p53 mutant SADA demonstrated 85-93% reductions in binding compared with GD2-SADA. Competition ELISA demonstrated comparable binding by all Ln-DOTA complexes (50% inhibition concentration, IC50 [nM]: Lu, 2.3; Tb, 1.0; Eu, 1.2; La, 1.9). TM-DOTA and empty DOTA showed negligible binding to GD2-SADA. In mouse SCLC models, median time to tumor volume for 10 mg/kg GD2-SADA with Lu-177-DOTA dosing at 32 mCi and 47 mCi/kg exceeded control groups by 30+ and 40+ days, respectively. Conclusions: The anti-GD2 domain of GD2-SADA mediated binding to GD2+ tumor cells, a highly avid interaction that required a functional p53 tetramerization domain. Selective binding of GD2-SADA to multiple Ln-DOTA complexes further supports the SADA platform’s theranostic applications. Negligible binding to TM-DOTA complexes or empty DOTA strongly suggests the absence of competitive antagonism, an important consideration for targeted delivery of the radioactive payload in patients. GD2-SADA Lu-177-DOTA showed potent and dose-responsive anti-tumor efficacy in mice. Clinical development of GD2-SADA with Lu-177-DOTA is underway in adults and adolescents with GD2+ tumors (Trial 1001).

KLF11 promotes the proliferation of breast cancer cells by inhibiting p53-MDM2 signaling

Abnormal Krüppel-like factor 11 (KLF11) expression is frequently found in tumor tissues and is associated with cancer prognosis, but its biological functions and corresponding mechanisms remain elusive. Here, we demonstrated that KLF11 functions as an oncoprotein to promote tumor proliferation in breast cancer cells. Mechanistically, at the transcription level, KLF11 decreased TP53 mRNA expression. Notably, KLF11 also interacted with and stabilized MDM2 through inhibiting MDM2 ubiquitination and subsequent degradation. This increase in MDM2 in turn accelerated the ubiquitin-mediated proteolysis of p53, leading to the reduced expression of p53 and its target genes, including CDKN1A, BAX, and NOXA1. Accordingly, data from animals further confirmed that KLF11 significantly upregulated the growth of breast cancer cells and was inversely correlated with p53 expression. Taken together, our findings reveal a novel mechanism for breast cancer progression in which the function of the tumor suppressor p53 is dramatically weakened.

Therapeutic targeting of TP53 nonsense mutations in cancer.

Mutations in the TP53 tumor suppressor gene occur with high prevalence in a wide range of human tumors. A significant fraction of these mutations (around 10%) are nonsense mutations, creating a premature termination codon (PTC) that leads to the expression of truncated inactive p53 protein. Induction of translational readthrough across a PTC in nonsense mutant TP53 allows the production of full-length protein and potentially restoration of normal p53 function. Aminoglycoside antibiotics and a number of novel compounds have been shown to induce full-length p53 in tumor cells carrying various TP53 nonsense mutations. Full-length p53 protein generated by translational readthrough retains the capacity to transactivate p53 target genes and trigger tumor cell death. These findings raise hopes for efficient therapy of TP53 nonsense mutant tumors in the future.

PKMYT1 Is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER+ Breast Cancer.

Endocrine therapies (ET) with cyclin-dependent kinase 4/6 (CDK4/6) inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of patient-derived xenografts (PDXs) from patients with 22 ER+ breast cancer demonstrated that protein kinase, membrane-associated tyrosine/threonine one (PKMYT1), a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX-derived organoids and PDXs, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.

The p53 protein - not only the guardian of the genome.

The p53 tumor suppressor protein is best known as an activator of cell cycle arrest and apoptosis. Only a fraction of p53-activated genes encode proteins affecting cellular replication and various forms of cell death (apoptosis, ferroptosis, autophagy). The p53-regulated genes can be divided into so-called the core transcriptional program, which comprises genes activated in most cell types by most activators, and into the group of genes activated in in cell- or stress-specific manner. Activation of p53 occurs via the extensive set of posttranslational modifications, which adjust its stability, interaction with other transcription regulators, and its ability to form a tetramer. Surprisingly, in mouse models, the activation of the best-studied p53 target genes encoding the inhibitor of the cell cycle (CDKN1A) or the inducers of apoptosis (e.g.NOXA,PUMA) is dispensable for protection against cancers. Thus, the non-classical functions of p53 must be studied to better understand its tumor suppressive mechanisms.