The potential role of circular RNAs in regulating p53 in different types of cancers.

P53 tumor suppressor is a major regulator of various cellular processes and functions. It has been reported that mutation or inactivation of p53 plays a crucial role in tumorigenesis in different types of cancers. Circular RNAs (circRNAs) are single-stranded non-coding RNAs that have significant post-transcriptional effects on the regulation of gene expression in various ways. These molecules can alter the expression and function of multiple genes and proteins. In the present study, we aimed to review circRNAs that regulate the expression, function, and stability of p53 and the possible interactions between these molecules and p53. Considering the importance of p53 in cancer and the network between p53 and circRNAs, future clinical trials targeting these circRNAs as therapeutic agents deserve worthy of attention.

Schisandrol B inhibits calcification of aortic valve by targeting p53 related inflammatory and senescence.

Calcific aortic valve disease (CAVD) primarily involves osteogenic differentiation in human aortic valve interstitial cells (hVICs). Schisandrol B (SolB), a natural bioactive constituent, has known therapeutic effects on inflammatory and fibrotic disorders. However, its impact on valve calcification has not been reported. We investigated the effect of SolB on osteogenic differentiation of hVICs. Transcriptome sequencing was used to analyze potential molecular pathways affected by SolB treatment. The study also included an in vivo murine model using aortic valve wire injury surgery to observe SolB's effect on valve calcification. SolB inhibited the osteogenic differentiation of hVICs, reversing the increase in calcified nodule formation and osteogenic proteins. In the murine model, SolB significantly decreased the peak velocity of the aortic valve post-injury and reduced valve fibrosis and calcification. Transcriptome sequencing identified the p53 signaling pathway as a key molecular target of SolB, demonstrating its role as a molecular glue in the mouse double minute 2 (MDM2)-p53 interaction, thereby promoting p53 ubiquitination and degradation, which further inhibited p53-related inflammatory and senescence response. These results highlighted therapeutic potential of SolB for CAVD via inhibiting p53 signaling pathway and revealed a new molecular mechanism of SolB which provided a new insight of theraputic mechanism for CAVD.

Progress in deciphering the role of p53 in diffuse large B-cell lymphoma: mechanisms and therapeutic targets.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, accounting for 30%-40% of non-Hodgkin lymphoma in adults. The mechanisms underlying DLBCL occurrence are extremely complex, and involve the B-cell receptor (BCR) and Toll-like receptor (TLR) signaling pathways, as well as genetic abnormalities and other factors. With the development of high-throughput sequencing, an increasing number of abnormal genes have been identified in DLBCL. Among them, the tumor protein p53 (TP53/p53) gene is important in DLBCL occurrence. Patients with DLBCL carrying TP53 gene abnormalities generally have poor prognosis and studies of p53 have potential to provide a better basis for their treatment. Normally, p53 is maintained at low levels through its interaction with murine double minute 2 (MDM2), and prevents tumorigenesis by mediating cell cycle arrest, apoptosis, and repair of damaged cells, among other processes. Therefore, the prognosis of patients with DLBCL harboring TP53 gene abnormalities (mutations, deletions, etc.) is poor, and targeting p53 for tumor therapy has become a research hotspot, following developments in molecular biology technologies. Current treatments targeting p53 mainly act by restoring the function or promoting degradation of mutant p53, and enhancing wild-type p53 protein stability and activity. Based on the current status of p53 research, exploration of existing therapeutic methods to improve the prognosis of patients with DLBCL with TP53 abnormalities is warranted.

Assessment of MDM2 Gene Locus Amplification by Fluorescence In-Situ Hybridization in Juvenile Ossifying Fibroma.

Juvenile ossifying fibroma (JOF) is an uncommon benign fibro-osseous lesion (BFOL) of the maxillofacial bones with a locally aggressive nature and a high recurrence rate. Murine Double Minute 2 (MDM2) is an oncogene located at chromosome 12 (12q13-15) that inhibits the tumor suppressor gene TP53. The presence of MDM2 gene locus amplification is a useful molecular adjunct in the evaluation of some sarcomas, including low-grade intramedullary osteosarcoma (LGIOS). JOF and LGIOS have some overlapping clinical and histopathological features. The aim of this study is to evaluate a series of JOF for the presence of MDM2 gene locus amplification using fluorescence in-situ hybridization (FISH). MATERIALS AND METHODS: With IRB approval, a search of the institutional files of the archives of the Oral Pathology and Surgical Pathology biopsy services at the University of Florida Health was performed. The cases were re-evaluated by an oral pathology resident, an oral and maxillofacial pathologist, and a bone and soft tissue pathologist. Cases with consensus in diagnosis were selected (n = 9) for MDM2 testing. Testing by FISH for MDM2 gene locus amplification was applied to all retrieved cases. RESULTS: The examined cases were all negative for MDM2 gene locus amplification via FISH testing. CONCLUSION: In our small series, JOF did not demonstrate MDM2 gene locus abnormality, a characteristic of LGIOS. This finding suggests that JOF has a distinct underlying pathogenesis. If confirmed in a larger series, these findings may be useful in distinguishing these two entities in cases with overlapping features or when minimal biopsy material is available.

A Case of Dedifferentiated Liposarcoma That Contributes to Accompanying Vessels of Various Size.

Dedifferentiated liposarcoma (DDLPS) is a non-lipogenic sarcoma, generally arising from well-differentiated liposarcoma (WDLPS), although it can develop de novo. DDLPS tumors rarely trans-differentiate into non-adipose mesenchymal tissues; however, the latter lack notable variety and mostly show striated muscle or osteogenic/chondrogenic differentiation. Here, we report a case of DDLPS that contained numerous atypical vessels. A man in his sixties presented with a large tumor in his right thigh, and the tumor was surgically resected. Microscopically, most of the tumor was WDLPS, but a minor portion showed DDLPS, consisting of high-grade spindle cells. Remarkably, the DDLPS contained vessels of various sizes with atypical cytoarchitecture, including vessels with seemingly muscular layers. Immunohistochemically, the atypical cells within the vascular wall expressed aSMA, consistent with smooth muscle cells or pericytes, whereas surrounding high-grade spindle cells only focally expressed it, and these aSMA-positive cells within the vessels exhibited MDM2 amplification by immuno-fluorescence in situ hybridization. Our results demonstrate that DDLPS can trans-differentiate into smooth muscle cells of various-sized accompanying vessels, which may support their survival and proliferation.

Case report: Atypical lipomatous tumor of the thigh in a four-year-old girl.

Atypical lipomatous tumors (ALTs) are locally aggressive adipocytic malignancies that frequently occur in middle-aged adults. We report the rare case of an ALT of the thigh that occurred in a 4-year-old girl. Since the tumor was initially diagnosed as a lipoblastoma by incisional biopsy, marginal resection was performed. Histopathological findings of the surgical specimen revealed the proliferation of mature and variously sized adipocytes, as well as ectopic ossification; these features differ from the typical findings of lipoblastoma. Immunohistochemical findings showed nuclear positivity for a murine double minute 2 (MDM2) and cyclin-dependent kinase 4 (CDK4) and negativity for pleomorphic adenoma gene 1 (PLAG1). Fluorescence in situ hybridization showed abnormal amplification of the MDM2 gene. The patient was thus finally diagnosed as having an ALT. No signs of local recurrence or metastasis were noted 1 year postoperatively. This case is instructive in the differential diagnosis of primary adipocytic tumors. Lipoblastomas are the most common adipocytic tumors in children, but if a tumor is located in the deep tissue or imaging findings are not typical, the possibility of ALT should be considered and immunohistochemistry for MDM2 and CDK4 should be added.

Discovery of PRMT3 Degrader for the Treatment of Acute Leukemia.

Protein arginine methyltransferase 3 (PRMT3) plays an important role in gene regulation and a variety of cellular functions, thus, being a long sought-after therapeutic target for human cancers. Although a few PRMT3 inhibitors are developed to prevent the catalytic activity of PRMT3, there is little success in removing the cellular levels of PRMT3-deposited ω-NG,NG-asymmetric dimethylarginine (ADMA) with small molecules. Moreover, the non-enzymatic functions of PRMT3 remain required to be clarified. Here, the development of a first-in-class MDM2-based PRMT3-targeted Proteolysis Targeting Chimeras (PROTACs) 11 that selectively reduced both PRMT3 protein and ADMA is reported. Importantly, 11 inhibited acute leukemia cell growth and is more effective than PRMT3 inhibitor SGC707. Mechanism study shows that 11 induced global gene expression changes, including the activation of intrinsic apoptosis and endoplasmic reticulum stress signaling pathways, and the downregulation of E2F, MYC, oxidative phosphorylation pathways. Significantly, the combination of 11 and glycolysis inhibitor 2-DG has a notable synergistic antiproliferative effect by further reducing ATP production and inducing intrinsic apoptosis, thus further highlighting the potential therapeutic value of targeted PRMT3 degradation. These data clearly demonstrated that degrader 11 is a powerful chemical tool for investigating PRMT3 protein functions.

A first-in-human phase I study of a novel MDM2/p53 inhibitor alrizomadlin in advanced solid tumors.

BACKGROUND: The mouse double minute 2 homolog (MDM2) oncogene exerts oncogenic activities in many cancers and represents a potential therapeutic target. This trial evaluated the safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of alrizomadlin (APG-115), a novel MDM2/p53 inhibitor, in patients with advanced solid tumors. PATIENTS AND METHODS: Patients with histologically confirmed advanced solid tumors who had progressed to standard treatment or lacked effective therapies were recruited. Alrizomadlin was administered once daily every other day for 21 days of a 28-day cycle until disease progression or intolerable toxicity. RESULTS: A total of 21 patients were enrolled and treated with alrizomadlin; 57.1% were male and the median age was 47 (25-60) years. The maximum tolerated dose of alrizomadlin was 150 mg and the recommended phase II dose was 100 mg. One patient in the 200-mg cohort experienced dose-limiting toxicity of thrombocytopenia and febrile neutropenia. The most common grade 3/4 treatment-related adverse events were thrombocytopenia (33.3%), lymphocytopenia (33.3%), neutropenia (23.8%), and anemia (23.8%). Alrizomadlin demonstrated approximately linear pharmacokinetics (dose range 100-200 mg) and was associated with increased plasma macrophage inhibitory cytokine-1, indicative of p53 pathway activation. Of the 20 assessable patients, 2 [10%, 95% confidence interval (CI) 1.2% to 31.7%] patients achieved partial response and 10 (50%, 95% CI 27.2% to 72.8%) showed stable disease. The median progression-free survival was 6.1 (95% CI 1.7-10.4) months, which was significantly longer in patients with wild-type versus mutant TP53 (7.9 versus 2.2 months, respectively; P < 0.001). Among patients with MDM2 amplification and wild-type TP53, the overall response rate was 25% (2/8) and the disease control rate was 100% (8/8). CONCLUSIONS: Alrizomadlin had an acceptable safety profile and demonstrated promising antitumor activity in MDM2-amplified and TP53 wild-type tumors. This study supports further exploration of alrizomadlin with recommended doses of 100 mg q.o.d. in 21 days on and 7 days off regimen.

Paratesticular Liposarcoma Presenting as an Inguinal Swelling: A Diagnostic Conundrum.

Paratesticular tumours are rare malignancies that are frequently misdiagnosed on presentation. We present a case of an elderly male with a six-month history of painless, progressively increasing left inguinal swelling. On preliminary examination and investigation, the swelling was misdiagnosed as a lymph nodal mass. Subsequently, a magnetic resonance imaging study detected a lesion that was not distinct from the spermatic cord. Biopsy testing of the said lesion was suggestive of poorly differentiated spindle cell neoplasm. The patient then underwent a high inguinal orchidectomy. Histopathological examination confirmed the diagnosis of a high-grade paratesticular dedifferentiated liposarcoma with rhabdomyoblastic differentiation. Due to the rarity of such tumours, the need for adjuvant chemotherapy and radiotherapy is debated.

Strong activation of p53 by actinomycin D and nutlin-3a overcomes the resistance of cancer cells to the pro-apoptotic activity of the FAS ligand.

The FAS ligand (FASLG) is expressed on lymphocytes, which employ it to activate death receptors on target cells. Cancer cells are generally resistant to apoptosis triggered by FASLG. In this work, we found a way to circumvent this resistance by treatment with actinomycin D (ActD) and nutlin-3a (Nut3a). We selected this drug combination based on our transcriptomic data showing strong activation of proapoptotic genes, including those for receptor-mediated apoptosis, in cells exposed to actinomycin D and nutlin-3a. To test our hypothesis, we pre-exposed cancer cell lines to this drug combination for 45 h and then treated them with recombinant FASLG. This almost instantaneously killed most cells. Actinomycin D and nutlin-3a strongly cooperated in the sensitization because the effect of the drugs acting solo was not as spectacular as the drug combination, which together with FASLG killed more than 99% of cells. Based on the caspase activation pattern (caspase-8, caspase-9, caspase-10), we conclude that both extrinsic and intrinsic pro-apoptotic pathways were engaged. In engineered p53-deficient cells, this pro-apoptotic effect was completely abrogated. Therefore, the combination of ActD + Nut3a activates p53 in an extraordinary way, which overcomes the resistance of cancer cells to apoptosis triggered by FASLG. Interestingly, other combinations of drugs, e.g., etoposide + nutlin-3a, actinomycin D + RG7112, and actinomycin D + idasanutlin had a similar effect. Moreover, normal human fibroblasts are less sensitive to death induced by ActD + Nut3a + FASLG. Our findings create the opportunity to revive the abandoned attempts of cancer immunotherapy employing the recombinant FAS ligand.

RNA Sequencing Reveals Candidate Genes and Pathways Associated with Resistance to MDM2 Antagonist Idasanutlin in TP53 Wild-Type Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a genetically and clinically diverse hematological cancer affecting middle-aged and elderly individuals. Novel targeted therapy options are needed for patients who relapse following initial responses or who are intrinsically resistant to current treatments. There is a growing body of investigation currently underway on MDM2 inhibitors in clinical trials, reflecting the increasing interest in including these drugs in cancer treatment regimens. One of the developed compounds, idasanutlin (RG7388), has shown promise in early-stage clinical trials. It is a second-generation MDM2-p53-binding antagonist with enhanced potency, selectivity, and bioavailability. In addition to the TP53 status, which is an important determinant of the response, we have shown in our previous studies that the SF3B1 mutational status is also an independent predictive biomarker of the ex vivo CLL patient sample treatment response to RG7388. The objective of this study was to identify novel biomarkers associated with resistance to RG7388. Gene set enrichment analysis of differentially expressed genes (DEGs) between RG7388-sensitive and -resistant CLL samples showed that the increased p53 activity led to upregulation of pro-apoptosis pathway genes while DNA damage response pathway genes were additionally upregulated in resistant samples. Furthermore, differential expression of certain genes was detected, which could serve as the backbone for novel combination treatment approaches. This research provides preclinical data to guide the exploration of drug combination strategies with MDM2 inhibitors, leading to future clinical trials and associated biomarkers that may improve outcomes for CLL patients.

The Strong Activation of p53 Tumor Suppressor Drives the Synthesis of the Enigmatic Isoform of DUSP13 Protein.

The p53 tumor suppressor protein activates various sets of genes depending on its covalent modifications, which are controlled by the nature and intensity of cellular stress. We observed that actinomycin D and nutlin-3a (A + N) collaborate in inducing activating phosphorylation of p53. Our recent transcriptomic data demonstrated that these substances strongly synergize in the upregulation of DUSP13, a gene with an unusual pattern of expression, coding for obscure phosphatase having two isoforms, one expressed in the testes and the other in skeletal muscles. In cancer cells exposed to A + N, DUSP13 is expressed from an alternative promoter in the intron, resulting in the expression of an isoform named TMDP-L1. Luciferase reporter tests demonstrated that this promoter is activated by both endogenous and ectopically expressed p53. We demonstrated for the first time that mRNA expressed from this promoter actually produces the protein, which can be detected with Western blotting, in all examined cancer cell lines with wild-type p53 exposed to A + N. In some cell lines, it is also induced by clinically relevant camptothecin, by nutlin-3a acting alone, or by a combination of actinomycin D and other antagonists of p53-MDM2 interaction-idasanutlin or RG7112. This isoform, fused with green fluorescent protein, localizes in the perinuclear region of cells.

Insights into the Interaction Mechanisms of Peptide and Non-Peptide Inhibitors with MDM2 Using Gaussian-Accelerated Molecular Dynamics Simulations and Deep Learning.

Inhibiting MDM2-p53 interaction is considered an efficient mode of cancer treatment. In our current study, Gaussian-accelerated molecular dynamics (GaMD), deep learning (DL), and binding free energy calculations were combined together to probe the binding mechanism of non-peptide inhibitors K23 and 0Y7 and peptide ones PDI6W and PDI to MDM2. The GaMD trajectory-based DL approach successfully identified significant functional domains, predominantly located at the helixes α2 and α2', as well as the ß-strands and loops between α2 and α2'. The post-processing analysis of the GaMD simulations indicated that inhibitor binding highly influences the structural flexibility and collective motions of MDM2. Calculations of molecular mechanics-generalized Born surface area (MM-GBSA) and solvated interaction energy (SIE) not only suggest that the ranking of the calculated binding free energies is in agreement with that of the experimental results, but also verify that van der Walls interactions are the primary forces responsible for inhibitor-MDM2 binding. Our findings also indicate that peptide inhibitors yield more interaction contacts with MDM2 compared to non-peptide inhibitors. Principal component analysis (PCA) and free energy landscape (FEL) analysis indicated that the piperidinone inhibitor 0Y7 shows the most pronounced impact on the free energy profiles of MDM2, with the piperidinone inhibitor demonstrating higher fluctuation amplitudes along primary eigenvectors. The hot spots of MDM2 revealed by residue-based free energy estimation provide target sites for drug design toward MDM2. This study is expected to provide useful theoretical aid for the development of selective inhibitors of MDM2 family members.

Synthesis, characterization, and anti-cancer potential of novel p53-mediated Mdm2 and Pirh2 modulators: an integrated In silico and In vitro approach.

Introduction: Leukemia is a global health concern that requires alternative treatments due to the limitations of the FDA-approved drugs. Our focus is on p53, a crucial tumor suppressor that regulates cell division. It appears possible to stabilize p53 without causing damage to DNA by investigating dual-acting inhibitors that target both ligases. The paper aims to identify small molecule modulators of Mdm2 and Pirh2 by using 3D structural models of p53 residues and to further carry out the synthesis and evaluation of hit candidates for anti-cancer potency by in vitro and in silico studies. Methods: We synthesized structural analogues of MMs02943764 and MMs03738126 using a 4,5-(substituted) 1,2,4-triazole-3-thiols with 2-chloro N-phenylacetamide in acetone with derivatives of PAA and PCA were followed. Cytotoxicity assays, including MTT, Trypan Blue Exclusion, and MTS assays, were performed on cancer cell lines. Anti-proliferation activity was evaluated using K562 cells. Cell cycle analysis and protein expression studies of p53, Mdm2, and Pirh2 were conducted using flow cytometry. Results: As for results obtained from our previous studies MMs02943764, and MMs03738126 were selected among the best-fit hit molecules whose structural analogues were further subjected to molecular docking and dynamic simulation. Synthesized compounds exhibited potent anti-proliferative effects, with PAC showing significant cytotoxicity against leukemia cells. PAC induced cell cycle arrest and modulated p53, Mdm2, and Pirh2 protein expressions in K562 cells. Molecular docking revealed strong binding affinity of PAC to p53 protein, further confirmed by molecular dynamics simulation. Discussion: The study presents novel anticancer compounds targeting the p53 ubiquitination pathway, exemplified by PAC. Future perspectives involve further optimization and preclinical studies to validate PAC's potential as an effective anticancer therapy.

Induction of the Mdm2 gene and protein by kinase signaling pathways is repressed by the pVHL tumor suppressor.

The tumor suppressor von Hippel-Lindau, pVHL, is a multifaceted protein. One function is to dock to the hypoxia-inducible transcription factor (HIF) and recruit a larger protein complex that destabilizes HIF via ubiquitination, preventing angiogenesis and tumor development. pVHL also binds to the tumor suppressor p53 to activate specific p53 target genes. The oncogene Mdm2 impairs the formation of the p53-pVHL complex and activation of downstream genes by conjugating nedd8 to pVHL. While Mdm2 can impact p53 and pVHL, how pVHL may impact Mdm2 is unclear. Like p53 somatic mutations, point mutations are evident in pVHL that are common in renal clear cell carcinomas (RCC). In patients with RCC, Mdm2 levels are elevated, and we examined whether there was a relationship between Mdm2 and pVHL. TCGA and DepMap analysis revealed that mdm2 gene expression was elevated in RCC with vhl point mutations or copy number loss. In pVHL reconstituted or deleted isogenetically match RCC or MEF cell lines, Mdm2 was decreased in the presence of pVHL. Furthermore, through analysis using genetic and pharmacological approaches, we show that pVHL represses Mdm2 gene expression by blocking the MAPK-Ets signaling pathway and blocks Akt-mediated phosphorylation and stabilization of Mdm2. Mdm2 inhibition results in an increase in the p53-p21 pathway to impede cell growth. This finding shows how pVHL can indirectly impact the function of Mdm2 by regulating signaling pathways to restrict cell growth.

Targeting MDM2-p53 interaction in Glioblastoma: Transcriptomic analysis and Peptide-Based inhibition strategy.

MDM2 is a gene that encodes a protein involved in cell survival, growth, and DNA repair. It has been implicated in the development and progression of glioblastoma (GBM). Inhibition of the MDM2-p53 interaction has emerged as a promising strategy for treating GBM. In this study, we performed comprehensive transcriptomic expression analysis from diverse datasets and observed MDM2 overexpression in a subset of GBM cases. MDM2 negatively regulates the major onco-suppressor p53. The interaction between MDM2 and p53 is a promising target for cancer therapy, as it can trigger p53-mediated cell death in response to different stress conditions, such as oncogene activation or DNA damage. In this study, we have identified a peptide-based inhibition of MDM2 as a therapeutic strategy for GBM. We have further validated the stability of the MDM2-peptide interaction using a molecular structural dynamics approach. The major trajectories, including root mean square of deviation (RMSD), root mean square of fluctuation (RMSF), and radius of gyration (RoG), indicate that the candidate peptides have a more stable binding compared to the native ligand and control drug. The stability of the binding interaction was further estimated by MMGBSA analysis, which also suggests that MDM2 has a stable binding with both peptide molecules. Based on these results, peptides P-1843 and P-3837 could be tested further for experimental validation to confirm their targeted inhibition of MDM-2. This approach could provide a highly selective and efficient inhibitor with potentially fewer side effects and less toxicity compared to small drug-based molecules.

Prognostic Factors for Long-Term Eribulin Response in a Cohort of Patients With HER2-Negative Metastatic Breast Cancer.

CONTEXT AND AIMS: Eribulin is used in taxane and anthracycline refractory HER2-negative metastatic breast cancers (MBC). Patients treated in pivotal clinical trials achieved low survival rates, therefore, the identification of prognostic criteria for long progression-free survival (PFS) is still an unmet medical need. In this study, we sought to determine potential prognostic criteria for long-term eribulin response in HER2-negative MBC. METHODS: Our retrospective cohort includes female patients with HER2-negative MBC treated with eribulin in Franche-Comté, France. We defined a long-term response as at least 6 months of eribulin treatment. The primary endpoint was the analysis of criteria that differ according to the progression-free survival. Secondary outcomes concerned overall survival and response rate. RESULTS: From January 2011 to April 2020, 431 patients treated with eribulin were screened. Of them, 374 patients were included. Median PFS was 3.2 months (2.8-3.7). Eighty-eight patients (23.5%) had a long-term response to eribulin. Four discriminant criteria allowed to separate PFS in 2 arms (PFS < 3 months or > 6 months) with a 78% positive predictive value: histological grade, absence of meningeal metastasis, response to prior chemotherapy, and OMS status. We have developed a nomogram combining these 4 criteria. Median overall survival was 8.5 months (7.0-9.5). CONCLUSION: Eribulin response in MBC can be driven by clinical and biological factors. Application of our nomogram could assist in the prescription of eribulin.

An MDM2 degrader shows potent cytotoxicity to MDM2-overexpressing acute lymphoblastic leukemia cells with minimal toxicity to normal cells/tissues.

The MDM2 oncogene is amplified and/or overexpressed in various human cancers and elevated expression of MDM2 protein acts as a survival factor promoting cancer progression through both p53-dependent and -independent pathways. Here, we report a novel small-molecule chemical compound (MX69-102) that we identified to induce MDM2 protein degradation, resulting in reactivation of p53, inhibition of XIAP, and potent cell growth inhibition and apoptosis in MDM2-overexpressing acute lymphoblastic leukemia (ALL) in vitro and in vivo. We have previously identified a compound (MX69) that binds to the MDM2 C-terminal RING domain and induces MDM2 protein degradation. In the present study, we performed structural modifications of MX69 and selected analog MX69-102, showing increased MDM2-targeting activity. MX69-102 exhibited significantly enhanced inhibitory and apoptotic effects on a group of MDM2-overexpressing ALL cell lines in vitro with IC50 values of about 0.2 µM, representing an approximately 38-fold increase in activity compared to MX69. MX69-102 also showed effective inhibition on xenografted human MDM2-overexpressing ALL in SCID mice. Importantly, MX69-102 had minimal or no inhibitory effect on normal human hematopoiesis in vitro and was very well tolerated in vivo in animal models. Based on the strong inhibitory and apoptotic activity against MDM2-overexpressing ALL, along with minimal or no toxicity to normal cells/tissues, MX69-102 is a candidate for further development as a novel MDM2-targeted therapeutic drug for refractory/MDM2-overexpressing ALL.

Establishment and characterization of a novel MDM2/MYCN-co-amplified neuroblastoma cell line, NBN-SHIM, established from a late recurrent stage MS tumor.

The biological heterogeneity of neuroblastoma underscores the need for an in vitro model of each molecularly defined subgroup to investigate tumorigenesis and develop targeted therapies. We have established a permanently growing cell line from a 12-year-old girl who developed a late recurrent stage MS, MDM2-amplified neuroblastoma arising in the liver and performed histological, molecular, cytogenetic, exome, and telomere analyses of the recurrent tumor and the cell line. On histology, the recurrent tumor was immunoreactive for TP53, CDKN1A, and MDM2. A molecular cytogenetic study of the recurrent tumor revealed the amplification of MDM2 but no amplification of MYCN. The established cell line, NBM-SHIM, showed amplification of both MDM2 and MYCN on double-minute chromosomes. A copy number evaluation based on exome data confirmed the finding for MYCN and MDM2 and further identified high ploidy on CDK4 and GLI2 loci in the recurrent tumor and the cell line. The telomere maintenance mechanism on the cell line is unusual in terms of the low expression of TERT despite MYCN amplification and alternative lengthening of telomeres suggested by positive value for C-circle assay and telomere contents quantitative assay. The cell line is unique because it was established from a MYCN-nonamplified, MDM2-amplified, late-relapsed stage MS neuroblastoma, and MYCN amplification was acquired during cell culture. Therefore, the cell line is a valuable tool for investigating neuroblastoma tumorigenesis and new molecular targeted therapies for disrupted ARF-TP53-MDM2 pathway and amplification of MDM2 and CDK4.

ESF1 positively regulates MDM2 and promotes tumorigenesis.

Eighteen S rRNA factor 1 (ESF1) is a predominantly nucleolar protein essential for embryogenesis. Our previous studies have suggested that Esf1 is a negative regulator of the tumor suppressor protein p53. However, it remains unclear whether ESF1 contributes to tumorigenesis. In this current research, we find that increased ESF1 expression correlates with poor survival in multiple tumors including pancreatic cancer. ESF1 is able to regulate cell proliferation, migration, DNA damage-induced apoptosis, and tumorigenesis. Mechanistically, ESF1 physically interacts with MDM2 and is essential for maintaining the stability of MDM2 protein by inhibiting its ubiquitination. Additionally, ESF1 also prevented stress-induced stabilization of p53 in multiple cancer cells. Hence, our findings suggest that ESF1 is a potent regulator of the MDM2-p53 pathway and promotes tumor progression.