Mouse Double Minute Research Articles

Tumor suppressor p53 mediates interleukin-6 expression to enable cancer cell evasion of genotoxic stress

The tumor suppressor p53 primarily functions as a mediator of DNA damage-induced cell death, thereby contributing to the efficacy of genotoxic anticancer therapeutics. Here, we show, on the contrary, that cancer cells can employ genotoxic stress-induced p53 to acquire treatment resistance through the production of the pleiotropic cytokine interleukin (IL)-6. Mechanistically, DNA damage, either repairable or irreparable, activates p53 and stimulates Caspase-2-mediated cleavage of its negative regulator mouse double minute 2 (MDM2) creating a positive feedback loop that leads to elevated p53 protein accumulation. p53 transcriptionally controls the major adenosine triphosphate (ATP) release channel pannexin 1 (Panx1), which directs IL-6 induction via a mechanism dependent on the extracellular ATP-activated purinergic P2 receptors as well as their downstream intracellular calcium (iCa2+)/PI3K/Akt/NF-ĸB signaling pathway. Thus, p53 silencing impairs Panx1 and IL-6 expression and renders cancer cells sensitive to genotoxic stress. Moreover, we confirm that IL-6 hampers the effectiveness of genotoxic anticancer agents by mitigating DNA damage, driving the expression of anti-apoptotic Bcl-2 family genes, and maintaining the migratory and invasive properties of cancer cells. Analysis of patient survival and relevant factors in lung cancer and pan-cancer cohorts supports the prognostic and clinical values of Panx1 and IL-6. Notably, IL-6 secreted by cancer cells during genotoxic treatments promotes the polarization of monocytic THP-1-derived macrophages into an alternative (M2-like) phenotype that exhibits impaired anti-survival activities but enhanced pro-metastatic effects on cancer cells as compared to nonpolarized macrophages. Our study reveals the precise mechanism for genotoxic-induced IL-6 and suggests that targeting p53-mediated IL-6 may improve the responsiveness of cancer cells to genotoxic anticancer therapy.

P02.23.A MDM2/X ALTERATIONS IN A MULTICOHORT RETROSPECTIVE ANALYSIS: A POTENTIAL TARGET OF TREATMENT IN PATIENTS WITH PRIMARY BRAIN TUMORS

Abstract BACKGROUND Primary brain tumors (pBTs) are a heterogeneous group of neoplasms with limited therapeutic alternatives. Disruption of the TP53 tumor suppressor pathway occurs in most high-grade gliomas. While TP53 loss-of-function mutations are poorly targetable, in 14% of patients this disruption is due to a Mouse double minute 2 homolog (MDM2) amplification. We aim to describe the characteristics of patients with MDM2 amplification and analyze its clinical and molecular factors using a cohort of patients with NGS data. MATERIAL AND METHODS Patients currently enrolled in the institutionally approved, retrospective multicenter study ESCAT-pBTs, which includes patients with pBT profiled used NGS methods (Foundation Medicine®, local NGS, Caris®, Oncomine®, copy number alterations (CNA), and fusion panels) were analyzed to determine the presence of MDM2 alterations (MDM2alt) and clinical factors including WHO 2021 diagnosis, sex, age, ECOG and other molecular findings. RESULTS A total of 493 pts with NGS and CNA were collected between Feb 2018 and January 2023 at 5 hospitals in Spain. The median age was 50.3 (range, 3.3-83.8), 14.2% had ECOG ≥2, 39.1% were women, 26.9% were 40 or younger, 21.5% had IDH1mut and 24.3% were methylated. The diagnosis of GBM (WHO 2021) was present in 68.8% of the patients and 33.8% had mutations or CNV in TP53. The overall prevalence of MDM2alt was 8.5%(42 pts), 92.9% gene amplifications, and 7,1% mutations. No differences were found in gender proportion between MDM2alt and no MDM2alt (Female: 37.1% vs 39.4% p:0.64) and no major variance in the percentage of ECOG ≥2 (16.6% vs 17.6% p:0.77). Prevalence in patients with GBM was: 11.8% vs 1.3% in patients with other pBTs. Concurrent mutations in MDM2alt patients were: CDK-4 copy number gain in 76,2%; a similar proportion of mutations in PI3KCA/PTEN was observed compared with the non-MDM2alt population in the pts with GBM (38,3% vs 50%, p:0.15). No significant variation was found in EGFR amplifications (42,9% vs 32% p:0.10) and EGFR mutations GBM MDM2 and no MDM2alt respectively (19% vs 18,7% p: 0.84). The median TMB was 5.22 (range from 2.24 to 13 mutations/megabase) in pts with MDM2alt and 4.76 (range from 1.26 to 53.7 mutations/megabase). 97,6% of the MDM2alt population was TP53 wild type. CONCLUSION MDM2alt is a common genomic event in patients with molecularly defined GBM and has a low prevalence in other pBT. The presence of MDM2alt is associated in our series with other genomic events such as PTEN loss, low TMB, and a high degree of CDK4 amplification. No statistically significant differences were found between the GBM MDM2alt and no MDM2alt subgroup in terms of molecular alterations and clinical aspects studied. These findings should inform the further clinical development of treatment strategies for MDM2alt pts.

Discovery of deoxyandrographolide and its novel effect on vascular senescence by targeting HDAC1.

Aconitum carmichaelii (Fuzi) is a traditional Chinese medicine that has been widely used in the clinic to save the dying life for over several thousand years. However, the medicinal components of Fuzi in treating vascular senescence (VS) and its potential mechanism remain unclear. In this study, a network pharmacology method was used to explore the possible components and further validated by experiments to get a candidate compound, deoxyandrographolide (DA). DA restrains aging biomarkers, such as p16, p21, γH2A.X, and p53 in vitro and in vivo blood co-culture studies. Histone deacetylase 1 (HDAC1), mouse double minute2 (MDM2), cyclin-dependent kinase 4, and mechanistic target of rapamycin kinase (mTOR) are predicted to be the possible targets of DA based on virtual screening. Subsequent bio-layer interferometry results indicated that DA showed good affinity capability with HDAC1. DA enhances the protein expression of HDAC1 in the angiotensin II-induced senescence process by inhibiting its ubiquitination degradation. Loss of HDAC1 by CRISPR/Cas9 leads to the disappearance of DA's anti-aging property. The enhancement of HDAC1 represses H3K4me3 (a biomarker of chromosomal activity) and improves chromosome stability. RNA sequencing results also confirmed our hypothesis. Our evidence illuminated that DA may achieve as a novel compound in the treatment of VS by improving chromosome stability.

KLF5/MDM2 Axis Modulates Oxidative Stress and Epithelial-Mesenchymal Transition in Human Lens Epithelial Cells: The Role in Diabetic Cataract

Diabetic cataract (DC) is a common cause of visual loss in older diabetic subjects. Krüppel-like factor 5 (KLF5) plays an essential role in migration and the epithelial-mesenchymal transition (EMT) in diverse cells and is involved in oxidative stress. However, the effects of KLF5 on DC remain unknown. This study aimed to examine the biological function of KLF5 in DC and its underlying mechanism. The expression patterns of KLF5 were detected in vivo and in vitro. Then, KLF5 was knocked down in human lens epithelial cells (HLECs) to explore its functional roles and underlying mechanisms. Dual-luciferase reporter assay and chromatin immunoprecipitation analysis were used to detect whether KLF5 could bind the promoter of E3 ubiquitin ligase mouse double minute 2 (MDM2), a key regulator of EMT. Lastly, the regulation of KLF5 in the biological behaviors of HLECs via MDM2 was analyzed. We found a significant increase of KLF5 in the DC lens anterior capsular, diabetic rat lens, and high glucose (HG)-stimulated HLECs. Knockdown of KLF5 inhibited oxidative stress, inflammation, migration, and EMT of HG-stimulated HLECs. KLF5 silencing impeded MDM2 expression and restricted the activation of MARK1/FAK and NF-κB signaling pathways in HLECs under HG condition. Additionally, KLF5 was found to bind the MDM2 promoter and enhance the transcriptional activity of MDM2. The protective effects by silencing KLF5 on HG-cultured HLECs could be offset by MDM2 overexpression. We demonstrated that knockdown of KLF5 alleviated oxidative stress, migration, and EMT of HG-cultured HLECs by regulating MDM2, suggesting a potential therapeutic strategy for DC.

Building on Foundations: Venetoclax-Based Combinations in the Treatment of Acute Myeloid Leukemia.

Frontline acute myeloid leukemia (AML) treatment is determined by a combination of patient and genetic factors. This includes patient fitness (i.e., comorbidities that increase the risk of treatment-related mortality) and genetic characteristics, including cytogenetic events and gene mutations. In older unfit patients, the standard of care treatment is typically venetoclax (VEN) combined with hypomethylating agents (HMA). Recently, several drugs have been developed targeting specific genomic subgroups of AML patients, enabling individualized therapy. This has resulted in investigations of doublet and triplet combinations incorporating VEN aimed at overcoming known resistance mechanisms and improving outcomes in older patients with AML. These combinations include isocitrate dehydrogenase-1/2 (IDH1/2) inhibitors (i.e., ivosidenib and enasidenib), fms-like tyrosine kinase 3 (FLT3) inhibitors (i.e., gilteritinib), anti-CD47 antibodies (i.e., magrolimab), mouse double minute-2 (MDM2) inhibitors, and p53 reactivators (i.e., eprenetapopt). This review summarizes ongoing trials aimed at overcoming known VEN resistance mechanisms and improving outcomes beyond that observed with HMA + VEN combinations in the treatment of AML.

MLF2 Negatively Regulates P53 and Promotes Colorectal Carcinogenesis.

Inactivation of the p53 pathway is linked to a variety of human cancers. As a critical component of the p53 pathway, ubiquitin-specific protease 7 (USP7) acts as a deubiquitinase for both p53 and its ubiquitin E3 ligase mouse double minute 2 homolog. Here, myeloid leukemia factor 2 (MLF2) is reported as a new negative regulator of p53. MLF2 interacts with both p53 and USP7. Via these interactions, MLF2 inhibits the binding of USP7 to p53 and antagonizes USP7-mediated deubiquitination of p53, thereby leading to p53 destabilization. Functionally, MLF2 plays an oncogenic role in colorectal cancer, at least partially, via the negative regulation of p53. Clinically, MLF2 is elevated in colorectal cancer and its high expression is associated with poor prognosis in patients with colorectal cancer. In wild-type-p53-containing colorectal cancer, MLF2 and p53 expressions are inversely correlated. These findings establish MLF2 as an important suppressor of p53 function. The study also reveals a critical role for the MLF2-p53 axis in promoting colorectal carcinogenesis.

Swietenolide isolated from Swietenia macrophylla King in Hook seeds shows in vitro anti-colorectal cancer activity through inhibition of mouse double minute 2 (MDM2) homolog

Swietenia macrophylla King in Hook (SM) is known to have several medicinal properties. Chloroform extracts of SM seeds (SMCE) as well as two isolated limonoids swietenine (1) and swietenolide (2) showed significant in vitro anti-CRC activity in human colon carcinoma (HCT116) cell line. 2 (IC50 = 5.6 µM) was found to be two times more potent than 1 (IC50 = 10 µM). Both compounds showed anti-CRC activity through inhibition of the Mouse Double Minute 2 homolog (MDM2) of the MDM2-p53 pathway. The Selectivity Index (S.I.) of isolated compounds 1 and 2 for cancer cells were about 6.6 and 12.8 fold respectively which was significantly better than the S.I. of the extract (S.I. ∼1.5).

Molecular pathways and gene networks in fetal hemoglobin as a novel protein target for beta-thalassemia

Abstract Background: Beta thalassemia is a condition in which the body cannot produce the beta subunit of hemoglobin due to harmful mutations in the globin gene that cause inadequate development of adult hemoglobin (HbA). In beta-thalassemic individuals, fetal hemoglobin (HbF), consisting of two and two subunits, is a potential replacement for HbA with significant therapeutic importance. HbF increase is a powerful and essential therapeutic tool to overcome the problem of beta-thalassemia. Materials and Methods: The GEO2R statistical tool and the GSE96060 dataset from the gene expression omnibus database were used to determine the differentially expressed genes (DEGs). The search tool for the retrieval of interacting genes program was used to reveal connections between these DEGs in samples, followed by applying the molecular complex detection algorithm to identify clusters of genes within these interaction networks. Using ClueGo and CluePedia, the discovered DEGs were subjected to functional annotation, including gene ontology (GO) and enriched molecular pathway analysis. Results: We searched the top 200 DEGs that met the criteria for significance (P-value 0.05; fold two change >1 or 1). Myeloid cell differentiation, erythrocyte differentiation, cellular detoxification, and heme binding are only a few examples of the biological processes and molecular pathways the GO analysis of DEGs identified as having significant alterations. The link between the DEGs in heme biosynthesis and transcriptional dysregulation in cancer was discovered by studying enriched Kyoto encyclopedia of genes and genomes pathways. To find possible targets for beta thalassemia treatments, we looked for the genes Krüppel-like factors 1 (KLF1) and mouse double minute 2 (MDM2). By activating both beta-globin and HbF gamma-globin genes, KLF1 encourages HbF repression, which is regulated by changing myeloblastosis expression. Conclusion: This study shows that the genes KLF1 and MDM2 are related to dysregulated molecular pathways, contribute to the development of beta thalassemia, and may be exploited as a platform for the induction of fetal hemoglobin in the development of therapeutics.

Hsa_circ_0023826 protects against glaucoma by regulating miR-188-3p/MDM4 axis.

Circular RNAs (circRNAs) are characterized as a class of covalently closed circRNA transcripts and are associated with various cellular processes and neurological diseases by sponging microRNAs. The most common feature of glaucoma, a form of retinal neuropathy, is the loss of retinal ganglion cells. Although the pathogenesis of glaucoma is not fully understood, elevated intraocular pressure is undoubtedly the only proven modifiable factor in the classic glaucoma model. This study investigated the role of circ_0023826 in glaucoma-induced retinal neurodegeneration by modifying the miR-188-3p/mouse double minute 4 (MDM4) axis. The expression pattern of circ_0023826 was analyzed during retinal neurodegeneration. The effect of circ_0023826, miR-188-3p, and MDM4 on retinal neurodegeneration in vivo was assessed by visual behavioral testing and HandE staining in glaucoma rats, while that on in vitro retinal ganglion cells (RGCs) was evaluated by MTT assay, flow cytometry, Western blot, and ELISA. Bioinformatics analysis, RNA pull-down assay, luciferase reporter assay were performed to reveal the regulatory mechanism of circ_0023826-mediated retinal neurodegeneration. Circ_0023826 expression was downregulated during retinal neurodegeneration. Upregulating circ_0023826 attenuated the visual impairment in rats and promoted the survival of RGCs in vitro. Circ_0023826 acted as a sponge of miR-188-3p sponge, resulting in increased expression of MDM4. MDM4 silencing or miR-188-3p upregulation reversed the protective effect of upregulated circ_0023826 on glaucoma-induced neuroretinal degeneration in vitro and in vivo. Overall, circ_0023826 protects against glaucoma by regulating the miR-188-3p/MDM4 axis, and targeted intervention of circ_0023826 expression is a promising therapeutic strategy for the treatment of retinal neurodegeneration.

P53/MDM2 Complex-Based Targeted Strategies in Colon Adenocarcinoma.

In the current molecular review, we describe the mechanisms of TP53/MDM2 deregulation and their impact on the colon adenocarcinoma molecular substrate and phenotype. Among the genes that are critically altered in carcinogenesis, the TP53 tumor suppressor gene is of major importance. The TP53 gene (gene locus: 17p13.1) regulates the cell cycle by controlling the G1/S and G2/M checkpoints securing the normal sequence of cell cycle phases. Furthermore, it is involved in apoptosis programmed cell death. The gene is mutated or epigenetically altered in all epithelial malignancies, including colon adenocarcinoma. Additionally, Mouse Double Minute 2 Homolog (MDM2), a proto-oncogene (12q14.3), acts as a major negative regulator for p53 expression in the p53-MDM2 auto-regulatory pathway. MDM2 binds directly to p53 and represses its transcriptional activity, promoting p53 degradation. CONCLUSION: In colon adenocarcinoma, MDM2 oncogene overexpression directly influences p53 oncoprotein expression levels.

A phase IIa/IIb, open-label trial of BI 907828, an MDM2–p53 antagonist, in patients with locally advanced/metastatic biliary tract carcinoma or pancreatic ductal adenocarcinoma: Brightline-2.

TPS4179 Background: Biliary tract cancer (BTC) and pancreatic ductal adenocarcinoma (PDAC) carry a median survival of one year in the advanced stages, and effective therapies are urgently needed. Mouse double minute 2 (MDM2) is amplified in ~5–6% of cases of BTC and ~1% of PDAC. MDM2, an E3 ubiquitin ligase, is an endogenous negative regulator of p53. Aberrations affecting MDM2 may drive oncogenesis, and indeed, preclinical as well as emerging clinical data suggest blocking MDM2 in TP53 wild-type tumors may be a potential therapeutic strategy. BI 907828 is an MDM2–p53 antagonist that binds to MDM2 and blocks its interaction with p53, thereby restoring p53 function, leading to cell-cycle arrest and apoptosis in TP53 wild-type tumor cells. In ongoing Phase I studies, BI 907828 has demonstrated initial signs of activity in selected advanced/metastatic solid tumors, including BTC and PDAC. Methods: Brightline-2 is a Phase IIa/IIb, open-label, single-arm, multicenter study (~60 sites) that aims to assess the efficacy, safety, and tolerability of BI 907828 monotherapy in patients with locally advanced, unresectable, or metastatic MDM2-amplified, TP53 wild-type BTC (adenocarcinoma histology; Cohort 1; n=~90) or PDAC (Cohort 2; n=~10) [NCT05512377]. Cohorts for other solid tumors may be added via protocol amendment. All patients will receive BI 907828 45mg orally once every 3 weeks. Key inclusion criteria include: ≥18 years of age; locally advanced/metastatic, histologically confirmed unresectable BTC/PDAC; progression or intolerance to standard therapies; local test indicating MDM2 amplification or MDM2 copy number ≥8 and TP53 wild-type (liquid biopsy not permitted); ≥1 measurable target lesion (RECIST v1.1); and ECOG PS 0/1. A key exclusion criterion is prior treatment with an MDM2–p53 antagonist. Treatment will continue until disease progression, unacceptable toxicity, or withdrawal of consent. The primary endpoint is objective response rate (ORR; based on blinded central independent review). Secondary endpoints are duration of response, progression-free survival, overall survival, disease control, occurrence of adverse events, and patient-reported health-related quality of life. In Phase IIa, an interim futility analysis will be performed after the initial 30 patients in Cohort 1 have either been followed for ≥12 weeks or have discontinued. If the cohort passes the non-binding interim futility boundary (ORR = 20%), the study will enter Phase IIb, and 60 additional patients will be enrolled onto Cohort 1, totaling 100 patients planned across Cohorts 1 and 2. The final primary analysis will be performed after all treated patients have been followed for ≥12 weeks or until study discontinuation. As of 13 December 2022, 1 patient has been enrolled. Clinical trial information: NCT05512377 .

A phase 2 study of alrizomadlin, a novel MDM2/p53 inhibitor, in combination with pembrolizumab for treatment of patients with malignant peripheral nerve sheath tumor (MPNST).

e14627 Background: MPNST is a rare and aggressive soft tissue malignancy accounting for 10% of all soft tissue sarcomas. This disease is characterized by high risk of local recurrence and distant metastasis, and there is no current effective standard of care treatment option for patients with MPNST, with the only curative option of complete resection. Therefore, further treatment options are warranted. Investigational mouse double minute 2 (MDM2) inhibitor alrizomadlin restores TP53 function, activating p53-mediated apoptosis in tumor cells. Alrizomadlin also functions as a host immunomodulator and may restore antitumor activity in patients with MPNST that progressed on PD-1/PD-L1 inhibitors. Methods: This US/Australian multicenter trial evaluated alrizomadlin combined with PD-1 inhibitor pembrolizumab in patients with MPNST that progressed on available therapy or in those for whom therapy was unavailable. ECOG performance status was between 0-2 for eligible patients. Alrizomadlin 150 mg was administered orally every other day for 2 consecutive weeks, with 1 week off, and pembrolizumab 200 mg intravenously for 30 minutes on Day 1 of a 21-day cycle. Results: As of December 12, 2022, preliminary clinical results are reported for 24 patients with MPNST, of whom 16 were male, with a median (range) age of 35 (14-73) years. Treatment (alrizomadlin or pembrolizumab)-related adverse events of any grade (>10%) were reported in 18 patients. They included nausea (41.7%), vomiting and anemia (37.5% each), fatigue and diarrhea (33.3% each), thrombocytopenia (25.0%), and decreased leukocyte and lymphocyte counts (12.5% each). A total of 12.5% of patients reported treatment-related serious adverse events, including colitis, abdominal pain, diarrhea, and pericardial effusion. Among17 efficacy evaluable patients, the clinical benefit rate, as defined by the combination of overall response rate and stable disease for ≥ 4 cycles, was 53% per Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v.1.1) and iRECIST. The median (range) treatment duration was 10 (4-20) cycles. Conclusions: Alrizomadlin combined with pembrolizumab is well tolerated and demonstrates meaningful clinical benefit in patients with MPNST for whom standard of care therapy is unavailable. Internal study identifiers: APG-115-US-002. Clinical trial information: NCT03611868 .

NOTOS: A pivotal study of navtemadlin, a first-in-class mouse double minute 2 inhibitor (MDM2i), in patients (pts) with TP53 wild-type (TP53WT) Merkel cell carcinoma (MCC) for whom anti PD-1/L1 therapy has failed.

TPS9600 Background: There are no approved therapies for pts with MCC for whom an anti PD-1/L1 agent has failed. The high rate of non-responsive disease after anti PD-1/L1 therapy together with high mortality rate and the absence of a standard of care in this setting highlight the urgent unmet need for effective novel therapies. Approximately 80% of MCC tumors carry the oncogenic Merkel cell polyoma virus (MCPyV) and most cases are TP53WT. Oncoproteins from MCPyV inhibit p53 tumor suppressor functions by activating MDM2. Navtemadlin, a potent, selective, orally available MDM2i, overcomes MDM2 dysregulation by restoring p53 activity and inducing apoptosis of TP53WT MCC tumors. Navtemadlin is the first targeted therapy to show single-agent activity in pts with metastatic MCC for whom anti-PD-1/L1 has failed (Wong ASCO 2022). In the dose finding study, evaluable pts (n=8) receiving the recommended phase 2 dose of navtemadlin (180 mg QD on Day 1-5/28-day cycle) demonstrated a 25% confirmed objective response rate (ORR), 38% unconfirmed + confirmed ORR, and 63% disease control rate. The median duration of response was not reached (range, 6-16.2+ months [mos]) and median time to treatment response was 4.1 mos (range, 1.2-7). Responses to navtemadlin were highest in those pts with no prior chemotherapy (chemo; Wong ASCO oral 2022). In pts receiving navtemadlin at doses of ≥180 mg, an ORR of 40% was reported with no prior chemo (n=15) versus 14% in those who received prior chemo (n=14, Wong ASCO oral 2022). Navtemadlin demonstrated an acceptable safety profile with the most common treatment emergent adverse events (TEAEs) cytopenias, nausea, vomiting, diarrhea, and fatigue. Grade 3/4 TEAEs were 32% anemia, 32% lymphopenia, and 19% thrombocytopenia (Wong 2022). Based on this encouraging data, the NOTOS study (navtemadlin induces p53-driven apoptosis in Merkel cell carcinoma) will evaluate the safety and efficacy of navtemadlin in two post PD-1/L1 treated cohorts: chemo-naive pts and chemo treated pts. Methods: Adults with MCC for whom anti PD-1/L1 therapy has failed, who have ≥1 measurable lesion per RECIST v1.1, ECOG PS 0-1 and TP53WT MCC by central testing will receive 180 mg QD on days 1-5 of a 28-day cycle until disease progression, unacceptable toxicity, death or withdrawal of consent. The primary endpoint is ORR per RECIST v1.1 by blinded independent review. Secondary endpoints include ORR per investigator, duration of response, progression-free survival, overall survival, clinical benefit rate (including stable disease for ≥10 wk, partial response or complete response), and safety. The NOTOS trial is currently enrolling (NCT03787602). Clinical trial information: NCT03787602 .

CCT6A and triple-negative breast cancer metastasis: Activating the AKT/MDM2/P53 pathway.

e13087 Background: Triple-negative breast cancer (TNBC) patients are prone to distant recurrence and poorer prognosis. The key molecules and underlying mechanisms of TNBC's aggressive phenotype remain to be further clarified. Methods: First, using the Kaplan-Meier Plotter ( https://kmplot.com/analysis ) and analyzing our collected clinical samples, we established the expression pattern and clinical value of the chaperonin containing TCP1 subunit 6A (CCT6A) in TNBC. We carried out the Cell Counting Kit-8 (CCK8), Transwell, and flow cytometry assay in the TNBC cell lines including MDA-MB-231 and BT549 to assess the effects of knockdown and overexpress of CCT6A. The mechanism of CCT6A in TNBC was identified by using the RNA-seq, KEGG analysis and Co-Immunoprecipitation Mass Spectrometry. Rescue assays verified the role of the AKT Serine/Threonine Kinase 1 (AKT) / Mouse double minute 2 homolog (MDM2)/P53 pathway on CCT6A. Then, the effect of CCT6A on tumor progression and metastasis in vivo was evaluated by the mouse models of tumorigenesis in situ and caudal artery lung metastasis. APG115, a pharmacological p53 activator and immune stimulatory MDM2 inhibitor, combined with anti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody Ipilimumab was found to prevent TNBC metastasis. Finally, we did a profiling of the tumor immune environment in mice. Results: CCT6A was highly expressed in TNBC ( p<0.0001), and patients with high expression of CCT6A had poor prognosis ( p=0.0053, HR=2.36 [1.27-4.39]). Of note, CCT6A was significantly associated with lymph node metastasis ( p<0.0001). CCT6A promoted cell proliferation, migration, and invasion while inhibiting cell apoptosis and achieving G1/S stage arrest through gain- and loss-of-function approaches. In terms of mechanism, Co-IP and Immunofluorescence assay verified that CCT6A interacted with Keratin 10 (KRT10) which can promote tumor metastasis. Compared with the control group, RNA-seq, and KEGG analysis suggested that AKT/MDM2/P53 signaling was inhibited after knockdown of CCT6A in MDA-MB-231. Furthermore, we found that the tumor volume and the number of lung metastasis sites were significantly reduced in the APG115 combined with Ipilimumab treatment group. Flow cytometry and multiple Immunofluorescence assay showed that TGF-β level from regulatory T cells decreased, while IFN-γ amount from CD8+T cells significantly increased with the combination (both p<0.05). Conclusions: Our research demonstrated that CCT6A can enhance metastasis of triple-negative breast cancer via up-regulating the AKT/MDM2/P53 pathway. These results provide rationales for MDM2 inhibitor combined with anti-CTLA-4 antibody for preventing TNBC metastasis.

A phase Ia/Ib, dose-escalation/expansion study of the MDM2–p53 antagonist BI 907828 in patients (pts) with solid tumors: Safety and efficacy in patients with dedifferentiated liposarcoma (DDLPS).

11554 Background: Inactivation of p53 is a key mechanism which promotes tumor survival and proliferation. p53 inactivation can be due to mutations in TP53 or downregulation of wild-type (wt) p53 by the key negative regulator mouse double-minute 2 (MDM2). BI 907828 is a highly potent MDM2–p53 antagonist which has demonstrated preclinical antitumor activity, particularly in TP53-wt MDM2-amplified DDLPS models. In this phase I study (NCT03449381), BI 907828 monotherapy is being evaluated in pts with advanced solid tumors, including DDLPS. During dose escalation (phase Ia), BI 907828 had a manageable safety profile and the selected recommended dose for expansion (RDE) was 45 mg q3w. Here we report safety data in all pts who received the RDE of 45 mg q3w and present efficacy data in the subgroup of pts with DDLPS. Methods: In Phase Ia, pts received one of two BI 907828 dosing schedules: Arm A, day 1 of 21-day cycles (q3w); Arm B, days 1 and 8 of 28-day cycles. During Phase Ib (dose expansion), pts were enrolled to Cohort 1 ( TP53wt, MDM2-amplified sarcoma) or Cohort 2 (other TP53wt, MDM2-amplified solid tumors). Here we focus on Cohort 1 (received BI 907828 45 mg q3w). The primary endpoint (phase Ib) was progression-free survival (PFS). Secondary endpoints/objectives included overall response rate (ORR) and grade ≥3 treatment-related AEs (TRAEs). Results: As of December 22, 2022, a total of 137 pts had been enrolled; 72 (52.6%) were male, 77 (56.2%)/59 (43.1%) had ECOG PS 0/1, and the median number of prior systemic therapies was 2 (range, 0–11). In the 73 pts who received the RDE of 45 mg q3w, the most common any-grade TRAEs were nausea (74.0%) and fatigue (61.6%). 33 (45.2%) pts had grade ≥3 TRAEs; the most common were thrombocytopenia (23.3%), neutropenia (21.9%), anemia (11.0%), and leukopenia (11.0%). 22 (30.1%) pts had TRAEs leading to dose reductions and 6 (8.2%) had TRAEs leading to treatment discontinuation. 20 pts (27.4%) had serious AEs; the most common were nausea, pulmonary embolism (each 4.1%), sepsis, small intestine obstruction, vomiting, thrombocytopenia, and pyrexia (each 2.7%). 50/73 pts who received 45 mg q3w had advanced DDLPS; all had MDM2-amplified disease. Of the 42 evaluable DDLPS pts, 8 achieved a confirmed PR (ORR 19.0%; locally assessed) and a further 29 achieved a best response of stable disease (including 2 with unconfirmed PR), giving a disease control rate of 88.1%. Preliminary median PFS was 8.1 months. Conclusions: BI 907828 demonstrated a manageable safety profile overall and encouraging preliminary efficacy was seen in pts with advanced MDM2-amplified DDLPS; Phase Ib is ongoing. BI 907828 is also being evaluated versus doxorubicin as first-line treatment for pts with advanced DDLPS in the ongoing Phase II/III Brightline-1 study (NCT05218499), for which the FDA has granted a Fast Track Designation. Clinical trial information: NCT03449381 .

Vitamin B6 regulates IL-33 homeostasis to alleviate type 2 inflammation.

Interleukin-33 (IL-33) is a crucial nuclear cytokine that induces the type 2 immune response and maintains immune homeostasis. The fine-tuned regulation of IL-33 in tissue cells is critical to control of the type 2 immune response in airway inflammation, but the mechanism is still unclear. Here, we found that healthy individuals had higher phosphate-pyridoxal (PLP, an active form of vitamin B6) concentrations in the serum than asthma patients. Lower serum PLP concentrations in asthma patients were strongly associated with worse lung function and inflammation. In a mouse model of lung inflammation, we revealed that PLP alleviated the type 2 immune response and that this inhibitory effect relied on the activity of IL-33. A mechanistic study showed that in vivo, pyridoxal (PL) needed to be converted into PLP, which inhibited the type 2 response by regulating IL-33 stability. In mice heterozygous for pyridoxal kinase (PDXK), the conversion of PL to PLP was limited, and IL-33 levels were increased in the lungs, aggravating type 2 inflammation. Furthermore, we found that the mouse double minute 2 homolog (MDM2) protein, an E3 ubiquitin-protein ligase, could ubiquitinate the N-terminus of IL-33 and sustain IL-33 stability in epithelial cells. PLP reduced MDM2-mediated IL-33 polyubiquitination and decreased the level of IL-33 through the proteasome pathway. In addition, inhalation of PLP alleviated asthma-related effects in mouse models. In summary, our data indicate that vitamin B6 regulates MDM2-mediated IL-33 stability to constrain the type 2 response, which might help develop a potential preventive and therapeutic agent for allergy-related diseases.

Regorafenib inhibits EphA2 phosphorylation and leads to liver damage via the ERK/MDM2/p53 axis

The hepatotoxicity of regorafenib is one of the most noteworthy concerns for patients, however the mechanism is poorly understood. Hence, there is a lack of effective intervention strategies. Here, by comparing the target with sorafenib, we show that regorafenib-induced liver injury is mainly due to its nontherapeutic target Eph receptor A2 (EphA2). EphA2 deficiency attenuated liver damage and cell apoptosis under regorafenib treatment in male mice. Mechanistically, regorafenib inhibits EphA2 Ser897 phosphorylation and reduces ubiquitination of p53 by altering the intracellular localization of mouse double minute 2 (MDM2) by affecting the extracellular signal-regulated kinase (ERK)/MDM2 axis. Meanwhile, we found that schisandrin C, which can upregulate the phosphorylation of EphA2 at Ser897 also has protective effect against the toxicity in vivo. Collectively, our findings identify the inhibition of EphA2 Ser897 phosphorylation as a key cause of regorafenib-induced hepatotoxicity, and chemical activation of EphA2 Ser897 represents a potential therapeutic strategy to prevent regorafenib-induced hepatotoxicity.

Histological and biochemical evaluation of the effects of silver nanoparticles (AgNps) versus titanium dioxide nanoparticles (TiO2NPs) on rat parotid gland

ABSTRACT The unlimited use of nanoparticles (NPs) results in toxic impacts on different tissues. The current study aimed to compare the adverse effects of AgNPs and TiO2NPs on the parotid gland of adult male albino rats as regards the histopathological, immunohistochemical, and biochemical changes, exploring the possible underlying mechanisms and the degree of improvement after cessation of administration. Fifty-four adult male albino rats were divided into control group (I), AgNPs-injected group (II), and TiO2NPs-injected group (III). We measured the levels of tumor necrosis factor-alpha (TNF-α) and interleukin (IL-6) in the serum, and levels of MDA and GSH in parotid tissue homogenate. Quantitative real-time polymerase-chain reaction (qRT-PCR) was used to measure the expression levels of peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC1-α), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), mouse double minute 2 (MDM2), Caspase-3 Col1a1, and Occludin. Parotid tissue sections were examined by light microscope (Hematoxylin & Eosin and Mallory trichrome stains), electron microscope, and immunohistochemical examination of CD68 and anti-caspase-3 antibodies. Both NPs severely affected the acinar cells and damaged the tight junction between them by enhancing expression of the inflammatory cytokines, inducing oxidative stress, and disturbing the expression levels of the studied genes. They also stimulated fibrosis, acinar cell apoptosis, and inflammatory cells infiltration in parotid tissue. TiO2NPs effects were less severe than AgNPs. Cessation of exposure to both NPs, ameliorated the biochemical and structural findings with more improvement in TiO2NPs withdrawal. In conclusion: AgNPs and TiO2NPs adversely affected the parotid gland, but TiO2NPs were less toxic than AgNPs.

Hyperhomocysteinemia activates NLRP3 inflammasome to cause hepatic steatosis and insulin resistance via MDM2-mediated ubiquitination of HSF1.

Hyperhomocysteinemia (HHcy) is associated with nonalcoholic fatty liver disease (NAFLD) and insulin resistance (IR). However, the underlying mechanism is still unknown. Recent studies have demonstrated that NLRP3 inflammasome activation plays a vital role in NAFLD and IR. Our study aimed to explore whether NLRP3 inflammasome contributed to HHcy-induced NAFLD and IR as well as dissected the underlying mechanism. C57BL/6 mice were fed a high-methionine diet (HMD) for 8weeks to establish the HHcy mouse model. Compared with a chow diet, HMD induced hepatic steatosis (HS) and IR as well as activation of hepatic NLRP3 inflammasome. Moreover, HHcy-induced NAFLD and IR characterization disclosed that NLRP3 inflammasome activation occurred in liver tissue of HMD-fed mice, but was very marginal in either NLRP3-/- or Caspase-1-/- mice. Mechanistically, high levels of homocysteine (Hcy) up-regulated the expression of mouse double minute 2 homolog (MDM2), which directly ubiquitinates heat shock transcription factor 1 (HSF1) and consequently activated hepatic NLRP3 inflammasome in vivo and in vitro. In addition, in vitro experiments showed P300-mediated HSF1 acetylation at K298 hindered MDM2-mediated ubiquitination of HSF1 at K372, which plays important role in determining the HSF1 level. Importantly, either inhibition of MDM2 by JNJ-165 or activation of HSF1 by HSF1A reversed HMD-induced hepatic NLRP3 inflammasome, and consequently alleviated HS and IR in mice. This study demonstrates that NLRP3 inflammasome activation contributes to HHcy-induced NAFLD and IR, and further identified that HSF1 as a new substrate of MDM2 and its decrease on MDM2-mediated ubiquitination at K372 modulates NLRP3 inflammasome activation. These findings may provide novel therapeutic strategies aimed at halting HS or IR.

Effect of Dan-Lou tablets on coronary heart disease revealed by microarray analysis integrated with molecular mechanism studies

Dan-Lou tablets (DLT) effectively treat coronary heart disease (CHD). However, its pharmacological mechanism in CHD treatment requires further investigation. This study aimed to elucidate the underlying pharmacological mechanisms of DLT in the treatment of CHD through clinical trials, microarray research, bioinformatics analysis, and molecular mechanism research. In this study, DLT improved coagulation function, endothelial injury, and levels of lipids, metalloproteases, adhesion molecules, inflammatory mediators, and homocysteine. The results of molecular biology research demonstrated that DLT can increase the gene and protein expressions of meningioma expressed antigen 5 (MGEA5) and mouse doubleminute 2 (MDM2) and inhibited the gene and protein expressions of signal transcription and transcription activator 5 B (STAT5B), tropomyosin-1 (TPM1), and aromatic hydrocarbon receptor nuclear transpose (ARNT). The results indicate that DLT reduced the extent of vascular endothelial damage in CHD rats by reducing the expressions of STAT5B, TPM1, and MDM2; inhibiting the inflammatory reaction; and increasing the expressions of ARNT and MGEA5.