MDMX Overexpression Research Articles

MiR-34c-5p inhibited fibroblast proliferation, differentiation and epithelial-mesenchymal transition in benign airway stenosis via MDMX/p53 pathway

Benign airway stenosis (BAS) means airway stenosis or obstruction that results from a variety of non-malignant factors, including tuberculosis, trauma, benign tumors, etc. In consideration of the currently limited research on microRNAs in BAS, this study aimed to explore the role and mechanism of miR-34c-5p in BAS. The expression of miR-34c-5p in BAS granulation tissues showed a significant down-regulation compared with the normal control group. Moreover, miR-34c-5p mimics suppressed the proliferation and differentiation of human bronchial fibroblasts (HBFs) and the epithelial-mesenchymal transition (EMT) of human bronchial epithelial cells (HBE). Conversely, miR-34c-5p inhibitors aggravated those effects. A dual-luciferase reporter assay confirmed that miR-34c-5p can target MDMX rather than Notch1. The over-expression of MDMX can reverse the inhibiting effect of miR-34c-5p on HBFs proliferation, differentiation and EMT. Furthermore, the expressions of tumor protein (p53) and PTEN were down-regulated following the over-expression of MDMX. In addition, the expressions of PI3K and AKT showed an up-regulation. In conclusion, miR-34c-5p was down-regulated in BAS and may inhibit fibroblast proliferation differentiation and EMT in BAS via the MDMX/p53 signaling axis. These findings expand the understanding of the role of miR-34c-5p and will help develop new treatment strategies for BAS.

Abstract 3929: MDMX overexpression in cancer cells confers significant resistance to MDMX inhibitor XI-006 and may modulate chemosensitivity through suppressing p53 activation of pro-apoptotic factors

Abstract MDM2 inhibition has gained popularity in recent years as a therapeutic strategy in targeting both solid tumors and hematological malignancies, and several small molecule inhibitors of the p53-MDM2 binding interface have progressed into clinical trials in recent years. Alongside direct stabilization of p53, MDM2 has further been identified to have immune implications in terms of CD8+ T-cell mediated anti-tumor immunity and checkpoint-blockade therapies. However, little work has been done to explore the therapeutic targeting potential of related protein MDMX (MDM4). Pan-cancer analyses conducted by our lab (in part presented at the ASCO 2022 Annual Meeting) identify an important role of MDMX in several tumor types. Particularly, MDMX amplification is associated with a higher risk of brain and liver metastasis, and MDMX amplification itself also presents with significant frequency (10%) in glioblastoma multiforme (GBM). Notably, MDMX amplification also served as a better predictor of reduced overall survival in NSCLC following checkpoint-blockade. We have also previously reported on associations between MDMX amplification and CDKN2A deep deletions in melanoma, a predictor of favorable response to checkpoint blockade. Such findings establish the need for further exploration to be done and the role of MDMX both in cell phenotype and potential immune regulation to be better characterized. Preliminary data acquired from transient overexpression experiments using lipofection of MDMX cDNA and MDMX inhibitor XI-006 (NSC207895) alone and in combination with chemotherapy further supports the viability of MDMX-inhibition as a therapeutic strategy in several cell lines. Overexpression of MDMX in HCT116 p53WT cells demonstrably confers remarkable resistance to MDMX inhibitor XI-006 at a 72-hour timepoint (IC50 of 104.75 uM in overexpressing cells versus 29.03 uM in WT), and treatment with oxaliplatin reveals both reduced basal and post-treatment expression of p53-upregulated modulator of apoptosis (PUMA). Further mechanistic workup of this phenomenon is underway, particularly with an emphasis on immuno-oncological therapeutic translations. Altogether, initial findings ultimately establish the need for further investigation into the mechanistic basis of MDMX-correlated clinical outcomes and the therapeutic potential of MDMX inhibition. Citation Format: Andrew George, Arielle De La Cruz, Shengliang Zhang, Ilyas Sahin, Praveen Srinivasan, Maximilian Schwermann, Morgan Turcotte, Taylor Arnoff, Wafik S. El-Deiry. MDMX overexpression in cancer cells confers significant resistance to MDMX inhibitor XI-006 and may modulate chemosensitivity through suppressing p53 activation of pro-apoptotic factors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3929.

MDMX elevation by a novel Mdmx–p53 interaction inhibitor mitigates neuronal damage after ischemic stroke

Mdmx and Mdm2 are two major suppressor factors for the tumor suppressor gene p53. In central nervous system, Mdmx suppresses the transcriptional activity of p53 and enhances the binding of Mdm2 to p53 for degradation. But Mdmx dynamics in cerebral infarction remained obscure. Here we investigated the role of Mdmx under ischemic conditions and evaluated the effects of our developed small-molecule Protein–Protein Interaction (PPI) inhibitors, K-181, on Mdmx–p53 interactions in vivo and in vitro. We found ischemic stroke decreased Mdmx expression with increased phosphorylation of Mdmx Serine 367, while Mdmx overexpression by AAV-Mdmx showed a neuroprotective effect on neurons. The PPI inhibitor, K-181 attenuated the neurological deficits by increasing Mdmx expression in post-stroke mice brain. Additionally, K-181 selectively inhibited HDAC6 activity and enhanced tubulin acetylation. Our findings clarified the dynamics of Mdmx in cerebral ischemia and provide a clue for the future pharmaceutic development of ischemic stroke.

1H, 15N and 13C backbone resonance assignments of the acidic domain of the human MDMX protein.

The human MDMX protein, also known as MDM4, plays a pivotal role in regulating the activity of the tumor suppressor protein p53 by restricting p53 transcriptional activity and stimulating the E3 ubiquitin ligase activity of another key regulatory protein, MDM2, to promote p53 degradation. MDMX is ubiquitously expressed in most tissue types and overexpression of MDMX has been implicated in many forms of cancer. MDMX has been shown to require an intact N-terminal p53-binding domain and C-terminal RING domain to exert inhibitory effects on p53. The presence of a tryptophan-rich sequence in the central acidic domain of MDMX has also been implicated in regulating the interaction between MDMX and p53, directly interacting with the p53 DNA-binding domain. To date, little structural information has been obtained for this acidic region of MDMX that encompasses the Trp-rich sequence. In order to gain insight into the structure and function of this region, we have carried out solution-state NMR spectroscopy studies utilizing the segment of MDMX spanning residues 181-300-with bounds specifically chosen through multiple sequence alignment-which encompasses nearly 25% of MDMX. Here, we report the 1H, 15N and 13C backbone chemical shift assignments of the acidic domain of MDMX and show that it exhibits hallmarks of intrinsic disorder and localized variation in inferred secondary structure propensity.

The ion channel TRPM7 regulates zinc-depletion-induced MDMX degradation

Zinc deficiency has been linked to human diseases, including cancer. MDMX, a crucial zinc-containing negative regulator of p53, has been found to be amplified or overexpressed in various cancers and implicated in the cancer initiation and progression. We report here that zinc depletion by the ion chelator TPEN or Chelex resin results in MDMX protein degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Restoration of zinc led to recovery of cellular levels of MDMX. Further, TPEN treatment inhibits growth of the MCF-7 breast cancer cell line, which is partially rescued by overexpression of MDMX. Moreover, in a mass-spectrometry-based proteomics analysis, we identified TRPM7, a zinc-permeable ion channel, as a novel MDMX-interacting protein. TRPM7 stabilizes and induces the appearance of faster migrating species of MDMX on SDS-PAGE. Depletion of TRPM7 attenuates, while TRPM7 overexpression facilitates, the recovery of MDMX levels upon adding back zinc to TPEN-treated cells. Importantly, we found that TRPM7 inhibition, like TPEN treatment, decreases breast cancer cell MCF-7 proliferation and migration. The inhibitory effect on cell migration upon TRPM7 inhibition is also partially rescued by overexpression of MDMX. Together, our data indicate that TRPM7 regulates cellular levels of MDMX in part by modulating the intracellular Zn2+ concentration to promote tumorigenesis.

MDMX is essential for the regulation of p53 protein levels in the absence of a functional MDM2 C-terminal tail

BackgroundMDM2 is an E3 ubiquitin ligase that is able to ubiquitinate p53, targeting it for proteasomal degradation. Its homologue MDMX does not have innate E3 activity, but is able to dimerize with MDM2. Although mouse models have demonstrated both MDM2 and MDMX are individually essential for p53 regulation, the significance of MDM2-MDMX heterodimerization is only partially understood and sometimes controversial. MDM2C462A mice, where the C462A mutation abolishes MDM2 E3 ligase activity as well as its ability to dimerize with MDMX, die during embryogenesis. In contrast, the MDM2Y487A mice, where the Y487A mutation at MDM2 C-terminus significantly reduces its E3 ligase activity without disrupting MDM2-MDMX binding, survive normally even though p53 is expressed to high levels. This indicates that the MDM2-MDMX heterodimerization plays a critical role in the regulation of p53. However, it remains unclear whether MDMX is essential for the regulation of p53 protein levels in the context of an endogenous MDM2 C-terminal tail mutation.ResultsHere, we studied the significance of MDM2-MDMX binding in an MDM2 E3 ligase deficient context using the MDM2Y487A mouse embryonic fibroblast (MEF) cells. Surprisingly, down-regulation of MDMX in MDM2Y487A MEFs resulted in a significant increase of p53 protein levels. Conversely, ectopic overexpression of MDMX reduced p53 protein levels in MDM2Y487A MEFs. Mutations of the RING domain of MDMX prevented MDMX-MDM2 binding, and ablated MDMX-mediated suppression of p53 protein expression. Additionally, DNA damage treatment and nuclear sequestration of MDMX inhibited MDMX activity to suppress p53 protein expression.ConclusionsThese results suggest that MDMX plays a key role in suppressing p53 protein expression in the absence of normal MDM2 E3 ligase activity. We found that the ability of MDMX to suppress p53 levels requires MDM2 binding and its cytoplasmic localization, and this ability is abrogated by DNA damage. Hence, MDMX is essential for the regulation of p53 protein levels in the context of an MDM2 C-terminal mutation that disrupts its E3 ligase activity but not MDMX binding. Our study is the first to examine the role of MDMX in the regulation of p53 in the context of endogenous MDM2 C-terminal mutant MEF cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

MDMX drives pre-leukaemia progression.

Ueda et al. report that overexpression of MDMX in pre-leukaemic stem cells provides these cells with a competitive advantage and promotes the progression of various pre-leukaemic conditions to acute myeloid leukaemia in several mouse models.

MDMX acts as a pervasive preleukemic-to-acute myeloid leukemia transition mechanism.

MDMX is overexpressed in the vast majority of patients with acute myeloid leukemia (AML). We report that MDMX overexpression increases preleukemic stem cell (pre-LSC) number and competitive advantage. Utilizing five newly generated murine models, we found that MDMX overexpression triggers progression of multiple chronic/asymptomatic preleukemic conditions to overt AML. Transcriptomic and proteomic studies revealed that MDMX overexpression exerts this function, unexpectedly, through activation of Wnt/β-Catenin signaling in pre-LSCs. Mechanistically, MDMX binds CK1α and leads to accumulation of β-Catenin in a p53-independent manner. Wnt/β-Catenin inhibitors reverse MDMX-induced pre-LSC properties, and synergize with MDMX-p53 inhibitors. Wnt/β-Catenin signaling correlates with MDMX expression in patients with preleukemic myelodysplastic syndromes and is associated with increased risk of progression to AML. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML transition mechanism in different genetically driven disease subtypes, and reveals Wnt/β-Catenin as a non-canonical MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

2003 – MDMX ACTS AS A PERVASIVE PRELEUKEMIC-TO-ACUTE MYELOID LEUKEMIA SWITCH MECHANISM

The p53 inhibitor MDMX is overexpressed in the vast majority of patients with AML; however, a causative role of MDMX in leukemogenesis has not yet been investigated. Mdmx transgenic mice (Mdmx-Tg) displayed increased number, proliferation and competitive advantage of hematopoietic stem cells compared to WT littermates. Crossing Mdmx-Tg with preleukemic mouse models representing frequent alterations in human AML, we generated three distinct compound models. We found that Mdmx overexpression triggered progression of each of the different chronic/asymptomatic preleukemic conditions to overt AML, and thereby markedly shortened survival. RNA sequencing revealed that Mdmx overexpression exerted this function through activation of Wnt/b-Catenin signaling in pre-LSC. Mechanistically, we found that MDMX bound CK1a, a canonical degrader of b-Catenin, and led to accumulation and nuclear translocation of b-Catenin. WNT/b-Catenin inhibitors or exogenous overexpression of CK1a were both effective in reversing Mdmx-induced pre-LSC properties, and showed further synergism in combination with MDMX inhibitor treatment. Competitive advantage and accumulation of b-Catenin upon Mdmx overexpression were still seen upon crossing into a p53-/- background, demonstrating that Mdmx-driven WNT/b-Catenin activation and its functional consequences are, at least part, independent of p53. Lastly, upregulation of WNT/b-Catenin pathways correlated with MDMX overexpression in large cohorts of patients with MDS and was associated with increased risk of progression to AML and poor survival. Our work identifies MDMX overexpression as a pervasive preleukemic-to-AML switch mechanism effective in different genetically-driven disease subtypes, and reveals WNT/b-Catenin as a novel, non-canonical p53-independent MDMX-driven pathway with therapeutic potential for progression prevention and cancer interception.

Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells

IntroductionMany human breast cancers overexpress the E3 ubiquitin ligase MDM2 and its homolog MDMX. Expression of MDM2 and MDMX occurs in estrogen receptor α-positive (ERα+) breast cancer and triple-negative breast cancer (TNBC). There are p53-independent influences of MDM2 and MDMX, and 80% of TNBC express mutant p53 (mtp53). MDM2 drives TNBC circulating tumor cells (CTCs) in mice, but the context-dependent influences of MDM2 and MDMX on different subtypes of breast cancers expressing mtp53 have not been determined.MethodsTo assess the context-dependent roles, we carried out MDM2 and MDMX knockdown in orthotopic tumors of TNBC MDA-MB-231 cells expressing mtp53 R280K and MDM2 knockdown in ERα+ T47D cells expressing mtp53 L194F. The corresponding cell proliferation was scored in vitro by growth curves and in vivo by orthotopic tumor volumes. Cell migration was assessed in vitro by wound-healing assays and cell intravasation in vivo by sorting GFP-positive CTCs by flow cytometry. The metastasis gene targets were determined by an RT-PCR array card screen and verified by qRT-PCR and Western blot analysis.ResultsKnocking down MDMX or MDM2 in MDA-MB-231 cells reduced cell migration and CTC detection, but only MDMX knockdown reduced tumor volumes at early time points. This is the first report of MDMX overexpression in TNBC enhancing the CTC phenotype with correlated upregulation of CXCR4. Experiments were carried out to compare MDM2-knockdown outcomes in nonmetastatic ERα+ T47D cells. The knockdown of MDM2 in ERα+ T47D orthotopic tumors decreased primary tumor volumes, supporting our previous finding that estrogen-activated MDM2 increases cell proliferation.ConclusionsThis is the first report showing that the expression of MDM2 in ERα+ breast cancer and TNBC can result in different tumor-promoting outcomes. Both MDMX and MDM2 overexpression in TNBC MDA-MB-231 cells enhanced the CTC phenotype. These data indicate that both MDM2 and MDMX can promote TNBC metastasis and that it is important to consider the context-dependent roles of MDM2 family members in different subtypes of breast cancer.

Abstract 4468: Repression of CD44 expression by p53 in a breast cancer progression model

Abstract CD44 is an important molecule in breast cancer. Although CD44 lacks its own signaling domain, it can associate with and co-stimulate signaling by a number of growth factor receptors, such as Her2 and EGF receptor 1. Knockdown of CD44 significantly reduces mammary tumor-initiating frequency and tumor weight. Disruption of tumor cell surface CD44 function induces apoptosis in metastatic mammary carcinoma cells in vivo. Some studies showed that the breast cancer stem cells are enriched in the CD44highCD24low population. The tumor suppressor p53 is mutated or functionally inactivated in over 70% of cancer. In breast cancer, p53 is mutated in ~35% cases, and ~50% breast cancers contain MDM2 or MDMX overexpression. A previous study showed that p53 repressed the expression of CD44 and that this activity was essential for the tumor-suppressor function of p53. We find that p53 potently represses the expression of CD44 in normal human breast epithelial cells. Mutation of the p53 binding site on the CD44 promoter greatly reduces the ability of p53 to repress CD44 expression. We show that p53 repression of CD44 expression is progressively reduced in a breast cancer progression model. Our ongoing studies are elucidating the underlying mechanisms. Our findings will help for better understanding and treatment of breast cancer. Citation Format: Fang Liu, Zhengxue Liu, Guannan Wang, Tanima Roy-Kundu, Isao Matsuura. Repression of CD44 expression by p53 in a breast cancer progression model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4468.

Abstract 5188: MDM2 and MDMX promote p53-independent initiation of circulating tumor cells from triple negative breast cancers

Abstract Many human breast cancers overexpress the E3 ubiquitin ligase MDM2 and its homolog MDMX. The Cancer Genome Atlas (TCGA) shows that high MDM2 and MDMX expression correlates with both estrogen receptor positive (ER+) and triple negative breast cancer (TNBC) subtypes. Overexpression of MDM2 and MDMX can drive breast cancer progression through molecular pathways that do not require wild-type p53 degradation. ER positive breast cancers often express wild-type p53 but 80% of TNBC express mutant p53 (mtp53). It has not been determined how MDM2 and MDMX expression in different sub-types of breast cancer, with mutant p53, influence proliferation and metastasis. We compared MDM2 and MDMX-dependent biological outcomes for mtp53 expressing ER+ T47D (mtp53 L194F) and TNBC cell type MDA-MB-231 (mtp53 R280K). In the ER+ cells no change was detected for real time cell migration or E-cadherin protein levels when MDM2 was knocked down by stable shRNA expression but cell proliferation was reduced. In support of this in vitro data, these MDM2 knockdown cells tested in an orthotopic xenograft model in NSG mice demonstrated no MDM2-mediated change in primary tumor invasiveness but did show a reduced tumor volume. Importantly, in the TNBC MDA-MB-231 cells, knocking down either MDM2 or MDMX did not reduce cell proliferation in vitro but greatly blocked cell migration. In support of this in vitro data, these MDA-MB-231 cells with either MDM2 or MDMX knockdown in NSG mice did not show a reduced primary tumor volume but showed a significant inhibition of the production of circulating tumor cells (CTCs). Our data shows that in different subtypes of breast cancers, MDM2 and MDMX can promote alternative p53-independent biological outcomes. The estrogen-activated MDM2 strongly promotes cell proliferation while MDM2 and MDMX overexpression in TNBC promotes circulating tumor cells and potentially metastasis. Understanding the alternative p53-independent roles of MDM2 and MDMX in ER+ and triple negative breast cancers will provide insight for the development of MDMs-targeted theranostics. Citation Format: Chong Gao, Gu Xiao, Ale Piersiga, Jiangtao Gou, Olorunseun Ogunwobi, Jill Bargonetti. MDM2 and MDMX promote p53-independent initiation of circulating tumor cells from triple negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5188.

A small molecule drug promoting miRNA processing induces alternative splicing of MdmX transcript and rescues p53 activity in human cancer cells overexpressing MdmX protein.

MdmX overexpression contributes to the development of cancer by inhibiting tumor suppressor p53. A switch in the alternative splicing of MdmX transcript, leading to the inclusion of exon 6, has been identified as the primary mechanism responsible for increased MdmX protein levels in human cancers, including melanoma. However, there are no approved drugs, which could translate these new findings into clinical applications. We analyzed the anti-melanoma activity of enoxacin, a fluoroquinolone antibiotic inhibiting the growth of some human cancers in vitro and in vivo by promoting miRNA maturation. We found that enoxacin inhibited the growth and viability of human melanoma cell lines much stronger than a structurally related fluoroquinolone ofloxacin, which only weakly modulates miRNA processing. A microarray analysis identified a set of miRNAs significantly dysregulated in enoxacin-treated A375 melanoma cells. They had the potential to target multiple signaling pathways required for cancer cell growth, among them the RNA splicing. Recent studies showed that interfering with cellular splicing machinery can result in MdmX downregulation in cancer cells. We, therefore, hypothesized that enoxacin could, by modulating miRNAs targeting splicing machinery, activate p53 in melanoma cells overexpressing MdmX. We found that enoxacin and ciprofloxacin, a related fluoroquinolone capable of promoting microRNA processing, but not ofloxacin, strongly activated wild type p53-dependent transcription in A375 melanoma without causing significant DNA damage. On the molecular level, the drugs promoted MdmX exon 6 skipping, leading to a dose-dependent downregulation of MdmX. Not only in melanoma, but also in MCF7 breast carcinoma and A2780 ovarian carcinoma cells overexpressing MdmX.Together, our results suggest that some clinically approved fluoroquinolones could potentially be repurposed as activators of p53 tumor suppressor in cancers overexpressing MdmX oncoprotein and that p53 activation might contribute to the previously reported activity of enoxacin towards human cancer cells.

Structural effects and competition mechanisms targeting the interactions between p53 and MDM2 for cancer therapy

Approximately half of all human cancers show normal TP53 gene expression but aberrant overexpression of MDM2 and/or MDMX. This fact suggests a promising cancer therapeutic strategy in targeting the interactions between p53 and MDM2/MDMX. To help realize the goal of developing effective inhibitors to disrupt the p53–MDM2/MDMX interaction, we systematically investigated the structural and interaction characteristics of p53 with inhibitors of its interactions with MDM2 and MDMX from an atomistic perspective using stochastic molecular dynamics simulations. We found that some specific α helices in the structures of MDM2 and MDMX play key roles in their binding to inhibitors, and that the hydrogen bond formed by the Trp23 residue of p53 with its counterpart in MDM2 or MDMX determines the dynamic competition processes of the disruption of the MDM2–p53 interaction and replacement of p53 from the MDM2–p53 complex in vivo. The results reported in this paper are expected to provide basic information for designing functional inhibitors and realizing new strategies of cancer gene therapy.

Development of cell‐penetrating peptide‐based drug leads to inhibit MDMX:p53 and MDM2:p53 interactions

The transcription factor p53 has a tumor suppressor role in leading damaged cells to apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2 and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53 interactions with nanomolar potency in vitro. With the aim of improving its cellular inhibitory activity, we produced a series of constrained pDI analogs featuring lactam staples that stabilize the bioactive helical conformation and fused them with a cell-penetrating peptide to increase cytosol delivery. We compared pDI and its analogs on their inhibitory potency, toxicity, and ability to enter cancer cells. Overall, the results show that these analogs keep their nanomolar affinity for MDM2 and MDMX and are highly active against cancer cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 853-863, 2016.

Increased Flexibility of Local Conformation in the N-Terminal Domain of MdmX Enhances Ligand-Binding Affinity

The overexpression of Mdm2 and MdmX ultimately impairs the cancer suppression of p53 function in approximately half of human cancers. Discovery of Mdm2 inhibitors has been made tremendous progress and some potent prodrugs have been considered for clinical investigation. Although MdmX is homologous to Mdm2 regarding their amino acid sequences and three-dimensional structures, the Mdm2 inhibitors exhibit weak affinity binding to MdmX. In order to aid in designing of MdmX inhibitors based on the Mdm2 inhibitor scaffolds, we engineered a disulfide bond to tie up the N-terminus and the C-terminus of the N-terminal domain of MdmX (N-MdmX) so that the pre-defined ligand-binding pocket on N-MdmX would be rigidified for docking an inhibitor. The resultant N-MdmX mutant was defined as N-MdmX2C and its interaction with nutlin-3a (a potent Mdm2 inhibitor) was characterized with isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD) and nuclear magnetic resonance (NMR) spectroscopy. The ITC data reveal that N-MdmX2C exhibits relatively-strong affinity binding to nutlin-3a compared to N-MdmX. The CD analysis indicates that both N-MdmX2C and N-MdmX have similar secondary structure content while their tertiary structures show difference. However, the 15N-1H HSQC NMR spectra reveal that the conformation of N-MdmX2C becomes more flexible compared to N-MdmX unexpectedly. Nevertheless, the 15N-1H HSQC NMR titration confirms that N-MdmX2C has higher binding affinity for nutlin-3a than N-MdmX. On the other hand, both N-MdmX2C and N-MdmX exhibit the same binding affinity to p53p investigated with ITC and NMR titration. Taken together, the current results suggest the confirmation of N-MdmX2C resembles a binding intermediate state for ligand binding, providing an ideal model for anticancer drug design targeting MdmX/Mdm2. (∗Corresponding authors)

Abstract C204: Characterization of TP53 mutations by DNA and RNA sequencing of platin-resistant ovarian cancer FFPE biopsies

Abstract Introduction: TP53 is the most frequently mutated gene across all cancers at approximately 50% and in cancers such as high grade serous ovarian up to 96%. In addition to the high incidence of DNA mutations at the gene locus, the TP53 pathway is also dysregulated through alternative mechanisms which involve other members of the regulatory cascade such as overexpression of Mdm2 or Mdmx. In contrast to other tumor-suppressors, TP53 possesses a spectrum of mutations including both loss and gain of function. Some mutations such as E177R result in a partial loss of TP53 function, eliminating the apoptotic fate while retaining senescence and cell-cycle arrest. Despite the centrality of TP53 in cancer, its prognostic significance remains unclear and the development of targeted therapies is ongoing. Characterizing TP53 mutation status in conjunction with transcriptional profiling of tumor biopsies will enable a more comprehensive understanding of the functional consequences of different classes of TP53 mutations and aid in distinguishing partial from total abrogation of TP53 pathway function. Asuragen has developed sensitive assays and algorithms to characterize DNA mutations and RNA expression profiles from the most challenging poor quality clinical samples. In order to enable a more complete understanding of the functional consequences of TP53 mutations (or lack thereof), we present an application of these approaches to characterize TP53 function in FFPE ovarian cancer biopsies through an integrated analysis of DNA and RNA sequencing. Methods: Primary FFPE tumor biopsies were collected from a cohort of 200 paclitaxel/carboplatin resistant ovarian cancer patients. DNA from isolated tumor biopsies and matched normal specimens was profiled by the Quantidex™ NGS TP53 Assay (Asuragen, Inc.) and library analysis was performed using the Quantidex™ Reporter. TP53 mutation status was independently assessed by the AmpliSeq™ Cancer Hotspot Panel (Thermo Fisher Scientific). Whole transcriptome RNA-Seq was performed on isolated total RNA material. Gene and isoform expression quantitation as well as SNV and indel calling was performed on the RNA-Seq libraries. Results: TP53 mutation status for Quantidex TP53 and AmpliSeq assays was found to be concordant for regions of TP53 covered by both assays. The Quantidex TP53 Assay has more complete coverage of COSMIC mutations, 99% compared to 68% for AmpliSeq. Many TP53 positives were detected by the Quantidex Assay but negative by AmpliSeq due to coverage differences. The remaining discordant calls in commonly covered regions were assessed with matched RNA-Seq data. The comparison with RNA-Seq, led to the observation of specific cases of allele specific preferential expression of TP53 gain of function mutations. RNA-Seq expression profiles were also evaluated leading to the identification of gene signatures that associate with different TP53 mutation classes. Conclusions: TP53 profiling through orthogonal DNA-Seq assays and matched RNA-Seq has enabled the identification of a set of high confidence associations between TP53 mutations and RNA expression signatures in ovarian cancer. These results point to a path for clinical assays in which DNA mutations and pathway signatures are integrated in support of diagnostic and precision medicine applications. Citation Format: Brian C. Haynes, Diane Ilsley, Marie Fahey, Gary J. Latham, Elizabeth B. Somers, Daniel J. O'Shannessy. Characterization of TP53 mutations by DNA and RNA sequencing of platin-resistant ovarian cancer FFPE biopsies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C204.

Retraction of "Efficient Reactivation of p53 in Cancer Cells by a Dual MdmX/Mdm2 Inhibitor".

The aberrant interaction between p53 and Mdm2/MdmX is an attractive target for cancer drug discovery because the overexpression of Mdm2 and/or MdmX ultimately impairs the function of p53 in approximately half of all human cancers. Recent studies have shown that inhibition of both Mdm2 and MdmX is more efficient than that of a single target in promoting cellular apoptosis in cancers. In this study, we demonstrate that a dual small-molecule antagonist of Mdm2/MdmX can efficiently reactivate the p53 pathway in model cancer cells overexpressing MdmX and/or Mdm2. The dual antagonist was rationally designed based on segmental mutational analysis of the N-terminal domain of MdmX and the crystal structure of the N-terminal domain of Mdm2 in complex with nutlin-3a (an Mdm2-specific inhibitor). The current work establishes a small molecule therapeutic candidate that targets cancers overexpressing Mdm2 and/or MdmX.

Caspase-dependent degradation of MDMx/MDM4 cell cycle regulatory protein in amyloid β-induced neuronal damage

MDMx/MDM4 is a negative regulator of the p53 tumor suppressor protein and is necessary for survival in dividing cells. MDMx is also expressed in postmitotic neurons, with prosurvival roles that are independent of its extensively described roles in carcinogenesis. We and others have shown a role for MDMx loss in neuronal death in vitro and in vivo in several neurodegenerative diseases. Further, we have recently shown that MDMx is targeted for proteolytic degradation by calcium-dependent proteases, calpains, in neurons in vitro, and that MDMx overexpression provided partial neuroprotection in a model of HIV-associated neurodegeneration. Here, we assessed whether amyloid β (Aβ)-induced MDMx degradation occurred in Alzheimer’s Disease (AD) models. Our data shows an age-dependent reduction in MDMx levels in cholinergic neurons within the cortex of adult mice expressing the swedish mutant of the amyloid precursor protein, APP in the Tg2576 murine model of AD. In vitro, Aβ treatment of primary cortical neurons led to the caspase-dependent MDMx degradation. Our findings suggest that MDMx degradation associated with neuronal death occurs via caspase activation in neurons, and that the progressive loss of MDMx protein represents a potential mechanism of Aβ-induced neuronal death during disease progression in AD.