E3 Ubiquitin Ligase MDM2 Research Articles

Regulation of the MDM2-p53 pathway by the ubiquitin ligase HERC2.

The p53 tumor suppressor protein is a transcription factor that plays a prominent role in protecting cells from malignant transformation. Protein levels of p53 and its transcriptional activity are tightly regulated by the ubiquitin E3 ligase MDM2, the gene expression of which is transcriptionally regulated by p53 in a negative feedback loop. The p53 protein is transcriptionally active as a tetramer, and this oligomerization state is modulated by a complex formed by NEURL4 and the ubiquitin E3 ligase HERC2. Here, we report that MDM2 forms a complex with oligomeric p53, HERC2, and NEURL4. HERC2 knockdown results in a decline in MDM2 protein levels without affecting its protein stability, as it reduces its mRNA expression by inhibition of its promoter activation. DNA damage induced by bleomycin dissociates MDM2 from the p53/HERC2/NEURL4 complex and increases the phosphorylation and acetylation of oligomeric p53 bound to HERC2 and NEURL4. Moreover, the MDM2 promoter, which contains p53‐response elements, competes with HERC2 for binding of oligomeric, phosphorylated and acetylated p53. We integrate these findings in a model showing the pivotal role of HERC2 in p53‐MDM2 loop regulation. Altogether, these new insights in p53 pathway regulation are of great interest in cancer and may provide new therapeutic targets.

DNA methylation modifier LSH inhibits p53 ubiquitination and transactivates p53 to promote lipid metabolism

BackgroundThe stability of p53 is mainly controlled by ubiquitin-dependent degradation, which is triggered by the E3 ubiquitin ligase MDM2. The chromatin modifier lymphoid-specific helicase (LSH) is essential for DNA methylation and cancer progression as a transcriptional repressor. The potential interplay between chromatin modifiers and transcription factors remains largely unknown.ResultsHere, we present data suggesting that LSH regulates p53 in cis through two pathways: prevention proteasomal degradation through its deubiquitination, which is achieved by reducing the lysine 11-linked, lysine 48-linked polyubiquitin chains (K11 and K48) on p53; and revival of the transcriptional activity of p53 by forming a complex with PKM2 (pyruvate kinase 2). Furthermore, we confirmed that the LSH–PKM2 interaction occurred at the intersubunit interface region of the PKM2 C-terminal region and the coiled-coil domains (CC) and ATP-binding domains of LSH, and this interaction regulated p53-mediated transactivation in cis in lipid metabolism, especially lipid catabolism.ConclusionThese findings suggest that LSH is a novel regulator of p53 through the proteasomal pathway, thereby providing an alternative mechanism of p53 involvement in lipid metabolism in cancer.

Insights into the Regulatory Roles of E3 Ubiquitin Ligases Associated with VHL-HIF Axis in Clear Cell Renal Cell Carcinoma

Renal cell carcinoma (RCC) accounts for up to 85% to 90% of all kidney cancers. Clear cell RCC (ccRCC), the major subtype of RCC, is mainly characterized by the inactivation of the tumor suppressor gene VHL. pVHL as an E3 ubiquitin ligase targets the hydroxylated form of HIF-α for proteasomal degradation. The loss of VHL function leads to HIF-α aggregation as the main mechanism of ccRCC. Recently, the regulations of ccRCC through other E3 ubiquitin ligases are emerging. Moreover, most of them are associated with the VHL-HIF axis. In this review, we mainly focus on seven E3 ubiquitin ligases JADE1, SIAH1, CHIP, FBXW7, MDM2, SPOP, and HAF. Based on reported researches of these ligases on ccRCC, they are divided into two groups: JADE1, SIAH1, CHIP, and FBXW7 that negatively regulate the growth of ccRCC; MDM2, SPOP, and HAF that promote ccRCC progression. In addition, we further verify these possible links between these E3 ligases and VHL-HIF axis in ccRCC based on The Cancer Genome Atlas RNA-seq and Clinical data. Understanding the mechanisms by which these ligases regulate ccRCC, especially the interplay between these ligases and VHL-HIF axis may enable the development of novel therapeutic approaches for ccRCC. Overall, the present review reveals the potential mechanism by which seven ligases (JADE1, SIAH1, CHIP, FBXW7, MDM2, SPOP, and HAF) regulate ccRCC progression in detail, especially their relationship to VHL-HIF axis.

In Vitro Analysis of Protein:Protein Interactions in the Human Cancer-Pertinent rp.eL42-p53-Mdm2 Pathway

Introduction: We have recently demonstrated that the eukaryote-specific large subunit ribosomal protein (rp) eL42 assists catalysis of peptide bond formation at the peptidyl transferase center of 80S ribosomes in eukaryotic cells. Recently, several ribosomal proteins were shown to have extraribosomal functions independent of protein biosynthesis. Such functions include regulation of apoptosis, cell cycle arrest, cell proliferation, neoplastic transformation, cell migration and invasion, and tumorigenesis through both Mdm2-p53-dependent and p53-independent mechanisms. Our objective is to demonstrate that overexpression of eL42 in tumor may incapacitate cell anti-tumor mechanism through interaction with the tumor suppressor protein p53 and its partner Mdm2. Methods: Co-immunoprecipitation technique and the binding assays on Biacore were used to probe interactions between recombinant eL42, p53 and Mdm2 proteins in a so-called rp-p53-Mdm2 axis. Results: We demonstrate that the ribosomal protein eL42, the tumor suppressor protein p53 and the ubiquitin E3 ligase Mdm2 interact with each other in a ternary rp.eL42:p53:Mdm2 complex. Precisely, the interaction between eL42 and p53 is characterized by a strong binding affinity (KD value in the nanomolar range) that is likely to trigger the sequestration of p53 and the inhibition of its tumor suppressor activity. Furthermore, the p53:Mdm2 and eL42:Mdm2 complexes exhibit comparable binding affinities in the micromolar range compatible with Mdm2 being the enzyme which ubiquitinates both the p53 and eL42 substrates. Interestingly, pyridoxal 5'-phosphate (PLP), one of the active forms of vitamin B6, binds to eL42 and significantly inhibits the interaction between eL42 and p53, in accordance with the observation that vitamin B6 is associated with reduced risk of cancer. Conclusion: Our study emphasized one more major mechanism of p53 downregulation involving its sequestration by eL42 upon the overexpression of this ribosomal protein. The mechanism described in the present report complemented the well-known p53 downregulation triggered by proteasomal degradation mediated through its ubiquitination by Mdm2.

Lipoic Acid Synergizes with Antineoplastic Drugs in Colorectal Cancer by Targeting p53 for Proteasomal Degradation.

Lipoic acid (LA) is a redox-active disulphide compound, which functions as a pivotal co-factor for mitochondrial oxidative decarboxylation. LA and chemical derivatives were shown to target mitochondria in cancer cells with altered energy metabolism, thereby inducing cell death. In this study, the impact of LA on the tumor suppressor protein p53 was analyzed in various colorectal cancer (CRC) cell lines, with a focus on the mechanisms driving p53 degradation. First, LA was demonstrated to trigger the depletion of both wildtype and mutant p53 protein in all CRC cells tested without influencing its gene expression and preceded LA-triggered cytotoxicity. Depletion of p53 coincided with a moderate, LA-dependent ROS production, but was not rescued by antioxidant treatment. LA induced the autophagy receptor p62 and differentially modulated autophagosome formation in CRC cells. However, p53 degradation was not mediated via autophagy as shown by chemical inhibition and genetic abrogation of autophagy. LA treatment also stabilized and activated the transcription factor Nrf2 in CRC cells, which was however dispensable for p53 degradation. Mechanistically, p53 was found to be readily ubiquitinylated and degraded by the proteasomal machinery following LA treatment, which did not involve the E3 ubiquitin ligase MDM2. Intriguingly, the combination of LA and anticancer drugs (doxorubicin, 5-fluorouracil) attenuated p53-mediated stabilization of p21 and resulted in synergistic killing in CRC cells in a p53-dependant manner.

Abstract 989: The relationship between TP53 gene status and carboxylesterase 2 expression in human gastric cancer

Abstract Carboxylesterases are serine hydrolases that are involved in the metabolisms of various endogenous and exogenous compounds. They are also required for activation of many anti-cancer prodrugs. For example, irinotecan (CPT-11), an anti-cancer prodrug that has been approved for the treatment of many types of solid tumors including gastric cancer, is converted by the carboxylesterase CES2 to its active metabolite 7-ethyl-10-hydroxycamptothesin (SN-38), a very potent topoisomerase I inhibitor. Among carboxylesterase isozymes, CES2 is most highly expressed in the gastrointestinal tract. Thus, the expression of CES2 may play an important role in local (i.e., intratumoral) activation of anti-cancer prodrugs such as irinotecan in the gut. Recent studies with cultured cancer cell lines have shown that CES2 expression is regulated by the tumor suppressor protein p53. However, whether CES2 expression is affected by the presence of p53 mutation in clinical cancer samples still remains unclear. In this study, we focused on the regulatory mechanism of CES2 expression in gastric cancer. First, we examined the relationship between TP53 gene status and CES2 expression using gastric cancer cell lines. Several gastric cancer cell lines expressing wild-type p53 (AGS, NUGC4, MKN74, and HSC58) were treated with nutlin-3a, a drug that inhibits the interaction between p53 and the E3 ubiquitin ligase MDM2 and thereby directly activates p53 signaling without genotoxic side effects. The expression of p21, a downstream target of p53, was increased following nutlin-3a treatment in two p53 wild-type cell lines NUGC4 and HSC58. The expression of CES2 was also upregulated by nutlin-3a in three p53 wild-type cell lines AGS, NUGC4, and HSC58. As expected, the expression levels of p21 and CES2 were not largely affected by nutlin-3a in gastric cancer cell lines with TP53 mutations. These results indicate that CES2 expression is positively regulated by the p53 pathway in most gastric cancer cells. We also investigated the relationship between TP53 gene status and CES2 expression in human gastric cancer samples. Our results may provide useful information for predicting the efficacy of anti-cancer prodrugs that are activated by CES2 in gastric cancer. Note: This abstract was not presented at the meeting. Citation Format: Yoshinori Kohira, Hyeon-Cheol Lee, Momoko Ishimine, Hajime Orita, Toshiyuki Kobayashi, Koichi Sato, Takehiko Yokomizo, Tetsu Fukunaga. The relationship between TP53 gene status and carboxylesterase 2 expression in human gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 989.

PS1437 SETD2 NON‐GENOMIC LOSS OF FUNCTION IN ADVANCED SYSTEMIC MASTOCYTOSIS (SM): PATHOGENETIC AND THERAPEUTIC IMPLICATIONS

Background:We have recently found that advanced SM patients (pts) and the HMC‐1.1 and −1.2 MCL cell lines display histone H3 Lys36 trimethylation (H3K36Me3) deficiency as a result of non‐genomic loss of function of the SETD2 methyltransferase. SETD2 plays a key role in the control of transcription and splicing fidelity and homologous recombination (HR). Inhibition of proteasome‐mediated degradation by bortezomib restored SETD2 protein expression and H3K36Me3.Aims:In this study, we aimed to identify the different players involved in SETD2 loss of function in ASM and MCL and to verify if they may be considered as promising therapeutic targets. Methods:Western blotting (WB) and immunofluorescence (IF) were used to assess proteins expression. Co‐immunoprecipitation assays were performed to investigate proteins interactions. Apoptosis and clonogenic assays were performed to test drugs efficacy.Results:Compared to cells from healthy donors, SETD2‐ and H3K36Me3‐deficient cell lines and pts had significantly higher levels of γH2AX and lower levels of Rad51. RNA‐seq in SETD2‐deficient pts showed evidence of transcription and splicing defects like transcription‐induced chimeras and intron retention not observed in healthy donors. Moreover we found that SETD2 co‐immunoprecipitates (co‐IP) with p53 in HMC‐1.1 and −1.2 cells. Rescuing the interaction between SETD2 and p53 with bortezomib stabilized p53 and upregulated the expression levels of p53 targets including p21, p27, Bax and Gadd45a. This restored inhibition of cell proliferation, block of the transition towards cell cycle checkpoints and consequent activation of apoptosis. The ubiquitin E3 ligase MDM2 was also found to complex with SETD2 after proteasomal inhibition. Treatment with the MDM2 inhibitor SP‐141 rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP‐141 treatment of HMC‐1 cells at micromolar doses induced cytostatic but not cytotoxic effects. Clonogenic assays supported the cytostatic effects of SP141 in HMC‐1.1 and −1.2 cells. siRNA‐mediated knock‐down of MDM2 also rescued SETD2 expression and activity, supporting the hypothesis that SETD2 hyper‐ubiquitination by MDM2 plays a role in SETD2 reduced stability and proteasomal degradation. Co‐IP also showed that SETD2 interacts with Aurora Kinase A (AKA), that is overexpressed in advanced SM. We found that AKA phosphorylates SETD2 and this phosphorylation may be involved in SETD2 loss of function. In fact, both inhibition by Danusertib and siRNA‐mediated silencing rescued SETD2 expression and activity. Finally, we performed clonogenic assays to evaluate the therapeutic potential of bortezomib, carfilzomib and ixazomib in neoplastic mast cells from 3 patients with advanced SM and we observed in all cases that both first and second generation inhibitors induced a significant reduction of clonogenic activity at nanomolar doses.Summary/Conclusion:AKA triggered MDM2‐mediated ubiquitination contributes to SETD2 non‐genomic loss of function in ASM and MCL. Loss of SETD2 and H3K36Me3 is associated with increased DNA damage and transcription and splicing defects in ASM and MCL pts and is likely to afford an alternative mechanism for the inactivation of the p53‐mediated checkpoint without the need for TP53 gene mutations. Inhibiting AKA or MDM2 activity or proteasome‐mediated degradation are promising therapeutic strategies. Supported by AIRC (project 16996) and AIL.

MDM2 promotes genome instability by ubiquitinating the transcription factor HBP1

Genome instability is a common feature of tumor cells, and the persistent presence of genome instability is a potential mechanism of tumorigenesis. The E3 ubiquitin ligase MDM2 is intimately involved in genome instability, but its mechanisms are unclear. Our data demonstrated that the transcription factor HBP1 is a target of MDM2. MDM2 facilitates HBP1 proteasomal degradation by ubiquitinating HBP1, regardless of p53 status, thus attenuating the transcriptional inhibition of HBP1 in the expression of its target genes, such as the DNA methyltransferase DNMT1 and histone methyltransferase EZH2, which results in global DNA hypermethylation and histone hypermethylation and ultimately genome instability. The repression of HBP1 by MDM2 finally promotes cell growth and tumorigenesis. Next, we thoroughly explored the regulatory mechanism of the MDM2/HBP1 axis in DNA damage repair following ionizing radiation. Our data indicated that MDM2 overexpression-mediated repression of HBP1 delays DNA damage repair and causes cell death in a p53-independent manner. This investigation elucidated the mechanism of how MDM2 promotes genome instability and enhances tumorigenesis in the absence of p53, thus providing a theoretical and experimental basis for targeting MDM2 as a cancer therapy.

Kinetic and thermodynamic effects of phosphorylation on p53 binding to MDM2

p53 is frequently mutated in human cancers. Its levels are tightly regulated by the E3 ubiquitin ligase MDM2. The complex between MDM2 and p53 is largely formed by the interaction between the N-terminal domain of MDM2 and the N-terminal transactivation (TA) domain of p53 (residues 15–29). We investigated the kinetic and thermodynamic basis of the MDM2/p53 interaction by using wild-type and mutant variants of the TA domain. We focus on the effects of phosphorylation at positions Thr18 and Ser20 including their substitution with phosphomimetics. Conformational propensities of the isolated peptides were investigated using in silico methods and experimentally by circular dichroism and 1H-NMR in aqueous solution. Both experimental and computational analyses indicate that the p53 peptides are mainly disordered in aqueous solution, with evidence of nascent helix around the Ser20-Leu25 region. Both phosphorylation and the phosphomimetics at Thr18 result in a decrease in the binding affinity by ten- to twenty-fold when compared to the wild-type. Phosphorylation and phosphomimetics at Ser20 result in a smaller decrease in the affinity. Mutation of Lys24 and Leu25 also disrupts the interaction. Our results may be useful for further development of peptide-based drugs targeting the MDM2/p53 interaction.

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.

Zinc deficiency causes neural tube defects through attenuation of p53 ubiquitylation.

Micronutrition is essential for neural tube closure, and zinc deficiency is associated with human neural tube defects. Here, we modeled zinc deficiency in mouse embryos, and used live imaging and molecular studies to determine how zinc deficiency affects neural tube closure. Embryos cultured with the zinc chelator TPEN failed to close the neural tube and showed excess apoptosis. TPEN-induced p53 protein stabilization in vivo and in neuroepithelial cell cultures and apoptosis was dependent on p53. Mechanistically, zinc deficiency resulted in disrupted interaction between p53 and the zinc-dependent E3 ubiquitin ligase Mdm2, and greatly reduced p53 ubiquitylation. Overexpression of human CHIP, a zinc-independent E3 ubiquitin ligase that targets p53, relieved TPEN-induced p53 stabilization and reduced apoptosis. Expression of p53 pro-apoptotic target genes was upregulated by zinc deficiency. Correspondingly, embryos cultured with p53 transcriptional activity inhibitor pifithrin-α could overcome TPEN-induced apoptosis and failure of neural tube closure. Our studies indicate that zinc deficiency disrupts neural tube closure through decreased p53 ubiquitylation, increased p53 stabilization and excess apoptosis.

A DHX9-lncRNA-MDM2 interaction regulates cell invasion and angiogenesis of cervical cancer

Cervical cancer (CC) is the third most common carcinoma and the fourth leading cause of cancer-associated mortality in women. Here, we report that MDM2-DHX9 interaction mediates CC motility and angiogenesis in a long noncoding RNA-dependent fashion. A long noncoding RNA, named lnc-CCDST, is significantly downregulated in CC tissues, and binds to pro-oncogenic DHX9. DHX9 is upregulated in CC tissue, and promotes CC cell motility and angiogenesis. The lnc-CCDST and DHX9 interaction promotes DHX9 degradation through the ubiquitin proteasome pathway. Furthermore, DHX9 bound to E3 ubiquitin ligase MDM2, and this interaction is enhanced by lnc-CCDST. Thus, lnc-CCDST promotes DHX9 degradation by serving as a scaffold to facilitate the formation of MDM2 and DHX9 complexes. Moreover, HPV oncogenes E6 and E7 abolish the expression of lnc-CCDST resulting in the increase of DHX9. Our results have revealed a novel mechanism by which high-risk HPVs promote motility and angiogenesis of CC by inhibiting expression of lnc-CCDST to disrupt MDM2 and DHX9 interaction, and DHX9 degradation, and identified a potential therapeutic target for CC.

MDM2 and Aurora Kinase a Contribute to SETD2 Loss of Function in Advanced Systemic Mastocytosis: Implications for Pathogenesis and Treatment

The SETD2 gene encodes the only methyltransferase responsible for histone H3 lysine 36 trimethylation (H3K36Me3) in humans. H3K36me3 play a key role in preserving the fidelity of transcription elongation and splicing. In addition, SETD2/H3K36me3 have more recently been implicated in the maintenance of genomic integrity by regulating homologous recombination (HR) repair, Mismatch Repair (MMR) mitotic spindle assembly and chromosome segregation. SETD2 deletions and/or inactivating mutations occur in many solid tumors and have recently been found also in acute leukemias. We have reported that the HMC-1.1 and -1.2 mast cell leukemia (MCL) cell lines and many advanced systemic mastocytosis (SM) patients (pts) display H3K36Me3 deficiency as a result of non-genomic loss of function of SETD2. Proteasome inhibition restored SETD2 protein expression and H3K36me3, suggesting that a functional protein is produced but rapidly degraded. In an attempt to uncover the mechanisms underlying this phenomenon, we used an in silico approach to identify candidate SETD2-interacting proteins, followed by experimental confirmation by co-immunoprecipitation (co-IP). We found that, after proteasomal inhibition, SETD2 co-immunoprecipitates with the ubiquitin E3 ligase MDM2. Treatment with the MDM2 inhibitor SP-141 rescued SETD2 expression and H3K36Me3, suggesting that MDM2 may play a role in SETD2 degradation in ASM and MCL. Moreover, SP-141 treatment of HMC-1 cells at micromolar doses induced cytostatic but not cytotoxic effects as shown by cell growth curves. Clonogenic assays supported the cytostatic effects of SP141 in HMC-1.1 and -1.2 cells. siRNA-mediated knock-down of MDM2 also rescued SETD2 expression and activity, further supporting the hypothesis that SETD2 hyper-ubiquitination by MDM2 plays a role in SETD2 reduced stability and proteasomal degradation. Co-IP also showed that SETD2 interacts with Aurora Kinase A, as it was suggested in silico. We found that Aurora A is overexpressed in advanced SM and may target SETD2 for phosphorylation. Both pharmacological inhibition by Danusertib and siRNA-mediated silencing of Aurora A rescued SETD2 expression and activity, raising the hypothesis that phosphorylation by Aurora A might be the trigger for MDM-2 mediated degradation of SETD2.To evaluate whether increased DNA damage and reduced HR proficiency can be observed in SETD2/H3K36Me3-deficient SM, we used western blotting (WB) and immunofluorescence (IF) to assess phosphorylated histone 2A.X (γH2AX) and Rad51 foci. Compared to cells from healthy controls, SETD2- and H3K36Me3-deficient cell lines and pts had significantly higher levels of γH2AX and lower levels of Rad51. RNA-seq in SETD2-deficient pts showed evidence of transcription and splicing defects like transcription-induced chimeras, intron retention and non-canonical splicing patterns not observed in healthy donors.Next, the ROSAD816V cell line, which displays SETD2 and H3K36me3 levels superimposable to healthy donors, was studied by WB and IF to assess γH2AX and Rad51 in steady state and after sub-lethal DNA damage by UV exposure. The same experiments were carried out after SETD2 silencing for 2 months. Cells with silenced SETD2 had significantly higher levels of γH2AX and were unable to activate the HR repair. Interestingly, clonogenic assays in ROSAD816V cells before and after SETD2 silencing showed that reduction of clonogenic potential after proteasomal or MDM2 inhibition is indeed SETD2-dependent (Figure 1A).Finally, we performed clonogenic assays to evaluate the therapeutic potential of bortezomib, carfilzomib and ixazomib in neoplastic mast cells from 3 patients with advanced SM and we observed in all cases that both first and second generation inhibitors induced a significant reduction of clonogenic activity at nanomolar doses (Figure 1B).Taken together, our results suggest that AKA and MDM2-mediated post-translational modifications contribute to SETD2 non-genomic loss of function in advanced SM. Loss of SETD2 and H3K36me3 is associated with increased DNA damage and transcription and splicing defects in patients. Inhibiting AKA or MDM2 activity or proteasome-mediated degradation are promising therapeutic strategies in patients with low SETD2 expression levels.Acknowledgments: study supported by AIRC (project code 16996) and AIL (Associazione Italiana contro le Leucemia, Linfomi e Mieloma). [Display omitted] DisclosuresBonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Pagano:Janssen: Speakers Bureau; Merck: Speakers Bureau; Gilead: Speakers Bureau; Basilea: Speakers Bureau; Pfizer: Speakers Bureau. Valent:Novartis: Honoraria; Pfizer: Honoraria; Incyte: Honoraria. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Soverini:Novartis: Consultancy; Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy.

Targeting the MTF2-MDM2 Axis Sensitizes Refractory Acute Myeloid Leukemia to Chemotherapy

Next generation sequencing of acute myeloid leukemia (AML) patient samples has enabled more granular risk stratification of patients; however, refractory AML patients can be found across all risk groups, suggesting that non-genetic lesions regulate chemoresponsiveness. Consistent with this hypothesis is the finding that many of the mutated AML driver genes are encode epigenetic modifiers. Thus, unraveling the epigenetic dysregulation in AML is critical to better understand disease initiation and progression, as well as develop targeted therapies. Metal Response Element Binding Transcription Factor 2/Polycomblike 2 (MTF2/PCL2) plays a fundamental role in recruiting the Polycomb repressive complex 2 (PRC2) to chromatin and we show that it is commonly silenced in primary AML patient cells at diagnosis. Furthermore, the loss of MTF2 in hematopoietic stem and progenitor cells (HSPCs) leads to an altered epigenetic state that underlies refractory AML. By implementing unbiased systems analyses, we identified the E3 ubiquitin ligase MDM2 that inhibits p53 as a direct target of MTF2-PRC2. MTF2 deficiency leads to over-expression of MDM2 and inhibition of p53-mediated cell cycle regulation and apoptosis, leading to chemoresistance and refractory AML. Targeting this dysregulated signaling pathway by MTF2 overexpression or MDM2 inhibitors sensitized refractory patient leukemic cells to induction chemotherapeutics and prevented relapse in AML patient-derived xenograft (PDX) mice. Therefore, we have uncovered a direct epigenetic mechanism by which MTF2 functions as a tumor suppressor required for AML chemotherapeutic sensitivity and identified a potential therapeutic strategy to treat refractory AML. DisclosuresSabloff:Celgene: Membership on an entity's Board of Directors or advisory committees.

Distinct control of PERIOD2 degradation and circadian rhythms by the oncoprotein and ubiquitin ligase MDM2.

The circadian clock relies on posttranslational modifications to set the timing for degradation of core regulatory components, which drives clock progression. Ubiquitin-modifying enzymes that target clock components for degradation mainly recognize phosphorylated substrates. Degradation of the circadian clock component PERIOD 2 (PER2) is mediated by its phospho-specific recognition by β-transducin repeat-containing proteins (β-TrCPs), which are F-box-containing proteins that function as substrate recognition subunits of the SCFβ-TRCP ubiquitin ligase complex. However, this mode of regulating PER2 stability falls short of explaining the persistent oscillatory phenotypes reported in biological systems lacking functional elements of the phospho-dependent PER2 degradation machinery. We identified PER2 as a previously uncharacterized substrate for the ubiquitin ligase mouse double minute 2 homolog (MDM2) and found that MDM2 targeted PER2 for degradation in a manner independent of PER2 phosphorylation. Deregulation of MDM2 plays a major role in oncogenesis by contributing to the accumulation of genomic and epigenomic alterations that favor tumor development. MDM2-mediated PER2 turnover was important for defining the circadian period length in mammalian cells, a finding that emphasizes the connection between the circadian clock and cancer. Our results not only broaden the range of specific substrates of MDM2 beyond the cell cycle to include circadian components but also identify a previously unknown regulator of the clock as a druggable node that is often found to be deregulated during tumorigenesis.

A novel uORF-based regulatory mechanism controls translation of the human MDM2 and eIF2D mRNAs during stress

Short upstream open reading frames (uORFs) are the most prevalent cis-acting regulatory elements in the mammalian transcriptome which can orchestrate mRNA translation. Apart from being “passive roadblocks” that decrease expression of the main coding regions, particular uORFs can serve as specific sensors for changing conditions, thus regulating translation in response to cell stress. Here we report a novel uORF-based regulatory mechanism that is employed under conditions of hyperosmotic stress by at least two human mRNAs, coding for translation reinitiation/recycling factor eIF2D and E3 ubiquitin ligase MDM2. This novel mode of translational control selectively downregulates their expression and requires as few as one uORF. Using a set of reporter mRNAs and fleeting mRNA transfection (FLERT) technique, we provide evidence that the phenomenon does not rely on delayed reinitiation, altered AUG recognition, ribosome stalling, mRNA destabilization or other known mechanisms. Instead, it is based on events taking place at uORF stop codon or immediately downstream. Functional aspects and implications of the novel regulatory mechanism to cell physiology are discussed.

A single synonymous mutation determines the phosphorylation and stability of the nascent protein.

p53 is an intrinsically disordered protein with a large number of post-translational modifications and interacting partners. The hierarchical order and subcellular location of these events are still poorly understood. The activation of p53 during the DNA damage response (DDR) requires a switch in the activity of the E3 ubiquitin ligase MDM2 from a negative to a positive regulator of p53. This is mediated by the ATM kinase that regulates the binding of MDM2 to the p53 mRNA facilitating an increase in p53 synthesis. Here we show that the binding of MDM2 to the p53 mRNA brings ATM to the p53 polysome where it phosphorylates the nascent p53 at serine 15 and prevents MDM2-mediated degradation of p53. A single synonymous mutation in p53 codon 22 (L22L) prevents the phosphorylation of the nascent p53 protein and the stabilization of p53 following genotoxic stress. The ATM trafficking from the nucleus to the p53 polysome is mediated by MDM2, which requires its interaction with the ribosomal proteins RPL5 and RPL11. These results show how the ATM kinase phosphorylates the p53 protein while it is being synthesized and offer a novel mechanism whereby a single synonymous mutation controls the stability and activity of the encoded protein.

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.

Gain-of-Function (GOF) Mutant p53 as Actionable Therapeutic Target.

p53 missense mutant alleles are present in nearly 40% of all human tumors. Such mutated alleles generate aberrant proteins that not only lose their tumor-suppressive functions but also frequently act as driver oncogenes, which promote malignant progression, invasion, metastasis, and chemoresistance, leading to reduced survival in patients and mice. Notably, these oncogenic gain-of-function (GOF) missense mutant p53 proteins (mutp53) are constitutively and tumor-specific stabilised. This stabilisation is one key pre-requisite for their GOF and is largely due to mutp53 protection from the E3 ubiquitin ligases Mdm2 and CHIP by the HSP90/HDAC6 chaperone machinery. Recent mouse models provide convincing evidence that tumors with highly stabilized GOF mutp53 proteins depend on them for growth, maintenance, and metastasis, thus creating exploitable tumor-specific vulnerabilities that markedly increase lifespan if intercepted. This identifies mutp53 as a promising cancer-specific drug target. This review discusses direct mutp53 protein-targeting drug strategies that are currently being developed at various preclinical levels.

Ubiquitin System

Ever since the discovery of ubiquitin in 1975[...].