bims-midomi 2025-06-15 papers

Int J Mol Sci. 2025 May 30. pii: 5286. [Epub ahead of print]26(11):

The Intrabody Against Murine Double Minute 2 via a p53-Dependent Pathway Induces Apoptosis of Cancer Cell.

Changli Wang, Wanting Liu, Haotian Guo, Tian Lan, Tianyi Wang, Bing Wang.

Murine double minute 2 (MDM2) is involved in various cancers and is an attractive target. The RING domain of MDM2 has been discussed as an alternative target to stabilize p53. Designing drugs to target the RING domain of MDM2 is an alternative approach to preventing MDM2-mediated deactivation of p53. In this study, we obtained a human VH single-domain antibody and revealed its regulatory effects and mechanisms. The RING domain of MDM2 was synthesized using a chemical synthesis method, and antibodies against the MDM2 RING domain were screened from a human VH single-domain antibody library and expressed intracellularly. A nuclear localization sequence was designed to ensure intrabody efficiency. The binding activity of the individually cloned antibodies was detected using ELISA. MTT and flow cytometry assays were used to detect the reactions related to intrabody in vitro. The combination and its influence on MDM2 were detected using immunoprecipitation assays, confocal microscopy, and Western blotting. The effects on apoptosis-related mitochondrial pathways downstream of p53 were examined using Western blotting. The influence on cell cycle distribution and cyclin-related proteins was detected using flow cytometry and Western blotting. A549 cell xenografts were constructed to assess the effect of intrabodies on growth in vivo. The molecular mechanisms of MDM2 and p53 were studied using Western blotting. Eight individual cloned antibodies were positive compared to the signals on the BSA-coated plates, especially intrabodies VH-HT3. In A549 and MCF-7 cell lines, VH-HT3 exhibited significant inhibitory effects on cell proliferation and apoptosis. VH-HT3 co-localized with MDM2 in the nucleus and cytoplasm. The specific combination of VH-HT3 triggered no significant effect on MDM2 activity for p53 degradation but upregulated the levels of factors downstream of p53, especially those in the mitochondrial apoptosis pathway. Moreover, VH-HT3 induced cell cycle arrest, and the expression of cyclin-related proteins was consistent with this observation. VH-HT3 also retarded the growth of A549 xenografts in vivo. Further tests suggested that VH-HT3 inhibited MDM2 function by increasing HIPK2 levels and activating p53 at the Ser46 site. VH-HT3, prepared from a human VH single-domain antibody library, inhibited p53 activity and produced a tumor-suppressive effect. The intrabody VH-HT3 is a candidate for the development of novel MDM2 inhibitors.

Keywords: MDM2; VH single-domain antibody; apoptosis; intrabody; p53

DOI: https://doi.org/10.3390/ijms26115286

J Med Chem. 2025 Jun 13.

MD-4251: A First-in-Class Oral MDM2 Degrader Inducing Complete Tumor Regression with Single-Dose Administration.

Ranjan Kumar Acharyya, Liyue Huang, Angelo Aguilar, Biao Hu, Longchuan Bai, Hoda Metwally, Donna McEachern, Wei Jiang, Yu Wang, Qiuxia Li, Bo Wen, Duxin Sun, Shaomeng Wang.

MDM2 is a key negative regulator of the tumor suppressor p53 and an attractive target for cancer therapy. We report the discovery of MD-4251, the first orally efficacious MDM2 degrader developed using PROTAC technology. MD-4251 induces potent and rapid MDM2 degradation in RS4;11 cells (DC50 = 0.2 nM; Dmax = 96% at 2 h), leading to robust p53 activation. It selectively inhibits the growth of acute leukemia cell lines with wild-type p53, with minimal activity in p53 mutant lines. MD-4251 shows excellent oral bioavailability in mice, favorable metabolic stability, and no CYP or hERG liabilities. A single oral dose induces sustained MDM2 depletion and attains complete tumor regression in vivo. These results support MD-4251 as a promising therapeutic candidate for cancers through depletion of MDM2.

DOI: https://doi.org/10.1021/acs.jmedchem.5c00809

iScience. 2025 Jun 20. 28(6): 112558

p21, ccng1, foxo3b, and fbxw7 contribute to p53-dependent cell cycle arrest.

Jun Wang, Zhang Li, Holly R Thomas, Ke Fan, Robert G Thompson, Yongjie Ma, David Crossman, Bradley K Yoder, John M Parant.

p53 is a transcription factor and important tumor suppressor gene, yet its mechanism of tumor suppression remains unclear. While PUMA/BBC3, NOXA/PMAIP1, and p21/CDKN1A regulate apoptosis and cell-cycle arrest, zebrafish lacking puma, noxa, and p21 do not develop cancer, suggesting additional p53 targets contribute to tumor suppression. We show that p53 can still induce cell-cycle arrest in the absence of p21, either following DNA damage or mdm2 loss, implicating other transcriptional target in p53-dependent cell-cycle arrest. We conducted a cross-species analysis to identify 137 conserved p53-upregulated genes. Our analysis also stresses the importance of ortholog to paralog analysis across species, since in many cases the paralog but not ortholog in differing species is p53 dependent. Using a CRISPR-Cas9 G0 "crispant" screen in mdm2, puma, noxa, and p21 quadruple knockout zebrafish, we identified ccng1, fbxw7, and foxo3b that are involved in p53-dependent cell-cycle arrest.

Keywords: Cell biology; Molecular biology; Omics

DOI: https://doi.org/10.1016/j.isci.2025.112558

Cancer Lett. 2025 Jun 06. pii: S0304-3835(25)00421-5. [Epub ahead of print]628 217854

Gobal crotonylome reveals that HNRNPC and its crotonylation promote p53-deficient tumor growth by stabilizing CCND1 and MCM3 mRNAs.

Liangjie Sun, Xiaolei Gao, Meng Wang, Yixin Zhang, Ruiqing Sun, Yang Chen, Yan Bai, Yi Li, Lan Luo, Chong Ding, Yixiang Wang.

The role of p53 deficiency or mutation in regulating nonhistone protein crotonylation and its impact on cancer development remain unclear. The present study identified crotonylation as a therapeutic target in patients with p53 deficiency or mutation in colorectal cancer. Crotonylome analysis revealed that p53 deficiency upregulated heterogeneous nuclear ribonucleoprotein C (HNRNPC) and HNRNPCK189Cr, promoting colorectal cancer cell proliferation by stabilizing CCND1 and MCM3 mRNAs through the MDM2/HDAC3 axis. Functional studies using HNRNPCK189Q (activating mutation) and HNRNPCK189R (inactivating mutation) confirmed the role of HNRNPCK189Cr in tumor growth. HDAC3 was identified as a specific decrotonylase of HNRNPCK189Cr. Sodium phytate, an HDAC3 agonist, effectively decrotonylated HNRNPCK189Cr and, in combination with HNRNPC siRNA, significantly inhibited the in vitro and in vivo growth of HCT116 p53-/- cells. An AOM/DSS-induced colorectal cancer model in K14-cre; p53 fl/fl mice validated the role of the p53/MDM2/HDAC3/HNRNPCK189Cr axis in tumor progression. Additionally, the findings in the oral cancer cells WSU-HN6 and non-small lung cancer cells H1299 were in line with those in the HCT116 cells, suggesting that the p53/MDM2/HDAC3/HNRNPCK189Cr regulatory mechanism plays a key role in a conserved manner. These findings revealed a novel mechanism by which p53 deficiency or mutation drives tumor progression via HNRNPCK189Cr through MDM2/HDAC3 axis-mediated CCND1 and MCM3 mRNA stability. Targeting HNRNPC and its crotonylation with sodium phytate and HNRNPC siRNA offers a promising therapeutic strategy, potentially converting p53 from an "undruggable" target to a "druggable" target.

Keywords: Crotonylation; HDAC3; HNRNPC; mRNA stability; p53 deficiency; p53 mutation

DOI: https://doi.org/10.1016/j.canlet.2025.217854

Front Pharmacol. 2025 ;16 1600960

Exploring the anti-gastric cancer mechanisms of Diosgenin through integrated network analysis, bioinformatics, single-cell sequencing, and cell experiments.

Zhangjun Yun, Qianru Yang, Chengyuan Xue, Yang Shen, Liyuan Lv, Suicai Mi, Li Hou.

Background: To comprehensively investigate the mechanism of action of Diosgenin elements against gastric cancer (GC).
Methods: Targets of Diosgenin were collected from six databases, and enrichment analysis was used to identify its associated diseases and biological pathways. GC-related genes were identified using weighted gene co-expression network analysis. A multi-approach strategy, including network analysis, bioinformatics, single-cell RNA sequencing, Mendelian randomization, and cell experiments, was used to explore the anti-GC mechanisms of Diosgenin.
Results: In this study, 605 Diosgenin targets were identified, with key involvement in cell apoptosis, TNF signaling, and platinum resistance pathways, demonstrating significant enrichment in GC. Diosgenin may exert its anti-GC effects through 311 targets, involving regulation of the cell cycle, p53, and FoxO signaling pathway. Key effectors, including CDK1, CCNA2, TOP2A, CHEK1, and PLK1, were identified. Single-cell sequencing indicated that TOP2A, HSP90AA1, and HSP90AB1 might be crucial immune regulatory targets of Diosgenin. Diosgenin significantly inhibited GC cell proliferation, colony formation, migration, and invasion. Evidence from western blot analysis indicates that Diosgenin exerts anti-GC effects by suppressing the expression of PLK1 and MDM2 proteins while upregulating p53 protein levels.
Conclusion: These findings highlight Diosgenin's potential as a promising therapeutic agent for GC, offering a foundation for future research and clinical applications.

Keywords: Diosgenin; MDM2; gastric cancer; mechanism; network analysis; p53; plk1

DOI: https://doi.org/10.3389/fphar.2025.1600960

Biotechnol Appl Biochem. 2025 Jun 08.

Identification of MDM4 as a Prognostic Biomarker and a Target for Therapeutics in Colorectal Cancer.

Xiao Shang, Xiaoqiang Zheng, Aimin Jiang, Ni Zhao, Yuru Chen, Na Liu, Min Chen, Xuan Liang, Zhiping Ruan, Tao Tian, Xiao Fu, Yu Yao.

Colorectal cancer (CRC) is a serious global health problem. Even with improvements in CRC diagnosis and treatment, many patients are diagnosed with metastatic disease, indicating the tumor has metastasized, and the survival rate for those with advanced CRC is still low. Immune checkpoint inhibitors (ICIs) have shown some promise for certain groups of CRC patients, specifically for those with mismatch repair deficiencies or microsatellite instability, but their overall effectiveness is still limited. Novel biomarkers and treatment targets are critically needed for the improvement of the diagnosis and treatment of CRC, ultimately improving patient outcomes. MDM4 (murine double minute 4) protein is important in controlling the tumor suppressor p53. MDM4 is similar in structure to MDM2 and is known to block p53's transcriptional ability, which can contribute to tumorigenesis. MDM4 is often found at higher levels in many cancers, including CRC, and has been linked to cancer progression through mechanisms that don't involve p53. However, MDM4's role in the tumor immune microenvironment of CRC remains unclear; its role in CRC prognosis and response to immunotherapy isn't fully understood. This study explores the biological, clinical, and immunological impact of MDM4 in CRC, focusing on its potential as a marker for prognosis and treatment target. This study is the first to comprehensively link MDM4 overexpression in CRC to immune evasion through reduced infiltration of CD8+ T cells and dendritic cells, establishing its role as an independent prognostic marker and a potential immunotherapy target. We explored the role of MDM4 in CRC by combining bioinformatic analyses and laboratory experiments. We gathered data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databasesWe performed Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA) to identify the key biological pathways linked to MDM4 in CRC. We also explored how MDM4 expression is associated with the immune microenvironment by examining the tumor-infiltrating lymphocytes in CRC tissues. Laboratory experiments were conducted to test the functional role of MDM4 in CRC cell lines. Our analysis showed that MDM4 expression was higher in CRC than in normal colorectal tissues, with even higher levels found in more advanced tumor stages. Increased MDM4 expression was linked to poorer progression-free survival (PFS) in CRC patients and was identified as an independent predictor of prognosis. Through pathway enrichment analyses, we found that MDM4 was involved in important tumor-related and immune pathways, including those regulating cell cycle progression and immune response. Notably, overexpression of MDM4 was associated with lower infiltration of CD8 T cells, natural killer (NK) cells, and dendritic cells in the tumor microenvironment, suggesting that MDM4 might help the tumor evade the immune system. In vitro experiments further confirmed these findings, showing that reducing MDM4 expression significantly slowed CRC cell growth and induced apoptosis. These results highlight the tumor-promoting role of MDM4 in CRC and suggest its possibility of becoming a therapeutic target. MDM4 is important in the progression and immune evasion of CRC. Its increased expression is implicated in disease progression and worse clinical outcomes, making it a valuable independent prognostic marker for CRC. Furthermore, MDM4's involvement in immune regulation, particularly in decreasing immune cell infiltration, suggests its potential as a target for immunotherapy. Targeting MDM4 could provide a new CRC treatment strategy, potentially improving patient outcomes by inhibiting tumor growth and boosting immune responses. Further research is needed to confirm MDM4 as a therapeutic target and to gain a deeper understanding of its function in CRC immunotherapy.

Keywords: colorectal cancer (CRC); immune microenvironment; murine double minute 4 (MDM4); prognosis

DOI: https://doi.org/10.1002/bab.70002