Nuclear Partner Research Articles

Nuclear pyruvate dehydrogenase complex regulates histone acetylation and transcriptional regulation in the ethylene response.

Ethylene plays its essential roles in plant development, growth, and defense responses by controlling the transcriptional reprograming, in which EIN2-C-directed regulation of histone acetylation is the first key step for chromatin to perceive ethylene signaling. But how the nuclear acetyl coenzyme A (acetyl CoA) is produced to ensure the ethylene-mediated histone acetylation is unknown. Here we report that ethylene triggers the accumulation of the pyruvate dehydrogenase complex (PDC) in the nucleus to synthesize nuclear acetyl CoA to regulate ethylene response. PDC is identified as an EIN2-C nuclear partner, and ethylene triggers its nuclear accumulation. Mutations in PDC lead to an ethylene hyposensitivity that results from the reduction of histone acetylation and transcription activation. Enzymatically active nuclear PDC synthesizes nuclear acetyl CoA for EIN2-C-directed histone acetylation and transcription regulation. These findings uncover a mechanism by which PDC-EIN2 converges the mitochondrial enzyme-mediated nuclear acetyl CoA synthesis with epigenetic and transcriptional regulation for plant hormone response.

Deciphering the Nuclear Role of Heme Oxygenase-1 in Prostate Cancer: Transcriptional Reprogramming and Neuroendocrine Differentiation

PURPOSE This study probes the role of Heme Oxygenase-1 (HO-1) and its nuclear interactors in prostate cancer (PCa). Previous results, demonstrated the anti-tumoral role of HO-1 in PCa and its nuclear translocation, hinting at its potential regulatory roles within the nucleus. Thus, we postulate that HO-1, alongside its interactors, can reprogram PCa cells to a less aggressive phenotype. METHODS From the 11 HO-1 nuclear interacting proteins previously identified, 4 were transcription factors Using ChIP Atlas we evaluated the binding affinity of these factors to their downstream gene targets. Gene ontology and KEGG pathway analyses were then performed to associate these targets with specific biological processes and pathologies. Additionally, RNA-seq data from patient-derived xenografts (PDXs) at MD Anderson Cancer Center (MDA PCa PDXs) were analyzed to evaluate the clinical relevance of these genes in the context of PCa heterogeneity. These PDXs offer a clinically relevant model due to their representation of the disease's heterogeneity. We scrutinized the expression of the gene clusters of interest in PDXs, including their association with neuroendocrine (NE) phenotypes. RESULTS The transcription factors among the HO-1 interactors previously described, were found to bind genes enriched in processes related to histone modification, acetylation, and transcription termination, implicating their role in gene expression regulation. KEGG pathways linked these genes to neurodegenerative diseases (including Parkinson's, Huntington's, and Alzheimer's disease), mirroring the neuroendocrine (NE) phenotype in advanced PCa, which typically emerges in advanced stages and is characterized by the transdifferentiating of epithelial cells into NE cells lacking androgen receptor expression. The unsupervised clustering of gene expression from PDXs showed that high expression of these genes is associated with an AR-negative NE histopathology, suggesting a potential for these interactors in prostate cancer subtyping. CONCLUSION HO-1 nuclear interactors, especially transcription factors, appear to influence gene transcription associated with NE differentiation and cancer aggressiveness. The therapeutic relevance of modulating HO-1's interaction with its nuclear partners warrants further investigation to ascertain its role in mitigating the NE of PCa.

El Presentation of the Multidimensional Couple: Socioemotional Impact Scale

Introduction: A systemic instrument is presented to measure the socioemotional network in relation to the partner and the person's perception of the impact of this intimate network on his or her partner for his or her classification. It is based on the idea that a nurtured social network brings positive benefits to one's nuclear partner. In order to verify this assumption both in research and in clinical practice, it is necessary to construct a complex instrument that allows reaching different dimensions within and outside the couple. Objective: The study seeks the construction and validation of the Multidimensional Couple scale to measure seven dimensions in the couple: emotional, cognitive, physical interest, protection, trust, respect and power, as well as an additional dimension to classify the type of couple. Method: An Exploratory Factor Analysis (EFA) and Exploratory Factor Analysis (CFA) were carried out to test the psychometric properties and the adequacy to the theoretical model. A total of 1149 people (71.5% women and 28% men) living in Mexico participated. Result: The presence of a scale formed by 7 dimensions in the couple and a second order factor is confirmed, which can be applied both by adjusting the answers to the couple itself and to other people different from the couple. The goodness-of-fit and reliability indices are satisfactory. Conclusion: This scale provides a psychometric instrument that allows the study of the relationship between the couple.

Abstract 5545: FADD activation renders hepatocellular carcinoma sensitive to immune checkpoint blockade by turning cold tumors hot

Abstract The heterogenicity of hepatocellular carcinoma (HCC) remains a key obstacle in turning the majority of ‘immune-cold’ tumors ‘hot’ for effective immune checkpoint blockade (ICB) therapy. Through studying the naturally-existed ‘hot’ HCC variant lymphoepithelioma-like (LEL)-HCC by RNA and whole exome sequencing analysis, we identified fas-associated death domain (FADD) as one of the top differentially expressed genes that associated with high tumoral CD8+T cell abundance. Of note, FADD upregulation was also observed in HCC patients with better ICB responsiveness. As FADD was mainly expressed in tumor cells from HCC by scRNAseq analysis, we constructed Fadd knockout stable cells using murine HCC cell lines. Interestingly, CRISPR knockout of tumoral Fadd led to increased tumor weights in immunocompetent but not immunodeficient mice, accompanied with decreased tumoral CD8+T cells. Mechanistically, FADD phosphorylation promoted NF-κB transcriptional activity to increase CCL5 secretion from HCC cells via interacting with its nuclear binding partners Src-associated-substrate-during-mitosis-of-68kDa/KH domain containing, RNA binding, signal transduction associated 1 (Sam68/KHDRBS1) and lysine (K)-specific methyltransferase 2D (KMT2D), thus directing CD8+T cell tumor infiltration. In addition, anti-PD1 triggered FADD phosphorylation via IFN-γ and TNF-ɑ release from CD8+T cells in ICB-sensitive but not resistant tumors. Most importantly, sequential FADD activation via genetic and pharmacologic approaches could re-sensitize the therapeutic efficacy of anti-programmed death 1 (PD1) antibody in ICB-resistant orthotopic and spontaneous HCC models by turning cold tumors hot. Taken together, our findings reveal a key molecular feature of ICB-responsive hot tumors that may guide the development of sequential combinatory immunotherapies for the majority of HCC patients with low responsiveness to ICB monotherapy. This project is supported by the University Grants Committee through General Research Fund (24110323, 14107622, 14104820) and the Health and Medical Research Fund (07180556). Citation Format: Jingying Zhou, Jiahuan Lu. FADD activation renders hepatocellular carcinoma sensitive to immune checkpoint blockade by turning cold tumors hot [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5545.

Desmin’s conformational modulation by hydrophobicity

Abstract Objectives Nucleocytoplasmic transport is one of the key features in regulation of cellular physiology. Developing a better understanding of the molecular mechanism underlying the nucleocytoplasmic shuttling of proteins can broaden our perspective and understanding on the elaborate sorting mechanisms within cells. Desmin is a muscle specific intermediate filament with amphiphilic properties and has interactions with the components of the nuclear pore complex which facilitates the transport between the cytoplasm and nucleus. The study aims to develop a better understanding of the amphiphilic nature of desmin and its relation to nucleocytoplasmic transport. Methods We conducted a proteomic analysis of desmin-immunoprecipitates to identify the nuclear partners of desmin. Additionally, we analysed the amphiphilic nature of desmin using a hydrophobicity assay to determine if it can undergo conformational changes to adapt to a hydrophobic environment. Results Using proteomic and in silico analysis we demonstrated that desmin interacts with several nups. The hydrophobicity assay results showed that desmin can increase its surface hydrophobicity in a hydrophobic environment. Conclusions Our findings suggest that desmin has the ability to undergo conformational changes under favourable conditions and possibly can be transported through nucleus via direct interaction with nups. Further analysis is required to understand the functional implications of this conformational change in vivo. Data are available via ProteomeXchange with identifier PXD047121.

Unique and overlapping roles of NRF2 and NRF1 in transcriptional regulation.

Transcription is regulated by specific transcription factors that mediate signaling in response to extrinsic and intrinsic stimuli such as nutrients, hormones, and oxidative stresses. Many transcription factors are grouped based on their highly conserved DNA binding domains. Consequently, transcription factors within the same family often exhibit functional redundancy and compensation. NRF2 (NFE2L2) and NRF1 (NFE2L1) belong to the CNC family transcription factors, which are responsible for various stress responses. Although their DNA binding properties are strikingly similar, NRF2 and NRF1 are recognized to play distinct roles in a cell by mediating responses to oxidative stress and proteotoxic stress, respectively. In this review, we here overview the distinct and shared roles of NRF2 and NRF1 in the transcriptional regulation of target genes, with a particular focus on the nuclear protein binding partners associated with each factor.

CERN-MEDICIS: Operational indicators to support the production of new medical radionuclides by mass-separation

CERN-MEDICIS is an isotope mass separation facility dedicated to biomedical research located in a type A work sector, receiving on average 50% of the 1.4 GeV protons delivered by the Proton Synchrotron Booster (PSB). It was commissioned with Radioactive Ion Beams (RIB’s) in 2017. MEDICIS has operated for the past 5 years in batch mode, with targets irradiated in a station located at the HRS beam dump, and with external sources provided by MEDICIS cyclotrons and nuclear reactors partners, notably during the Long Shutdown (LS2). Additional features of the facility include the MELISSA laser ion source, radiochemistry on implanted radionuclides and an online gamma-ray spectroscopy implantation monitoring. In 2022, we introduced Key Performance Indicators (KPI’s) to monitor the operation of the facility for collected efficiencies, the optimisation of the radiological risks and evaluate impact of possible modifications of the station, paralleling for instance LHC’s integrated luminosity. Defined KPI’s cover aspects in the operation cycle, e.g. planning in CERN schedule, target irradiations, duration of the process, radiological risk mitigation, facility up-time, developments and maintenance. MEDICIS KPI’s can help distinguish which of the operation and infrastructure life cycle requires immediate intervention, developments or consolidation. Those are related to the irradiation stations and irradiation possibilities, the beamlines (parallel collections), target and ion sources (reliability), robot handling and infrastructure, or the separation process itself.

The Identification of Nuclear FMRP Isoform Iso6 Partners.

A deficiency of FMRP, a canonical RNA-binding protein, causes the development of Fragile X Syndrome (FXS), which is characterised by multiple phenotypes, including neurodevelopmental disorders, intellectual disability, and autism. Due to the alternative splicing of the encoding FMR1 gene, multiple FMRP isoforms are produced consisting of full-length predominantly cytoplasmic (i.e., iso1) isoforms involved in translation and truncated nuclear (i.e., iso6) isoforms with orphan functions. However, we recently implicated nuclear FMRP isoforms in DNA damage response, showing that they negatively regulate the accumulation of anaphase DNA genomic instability bridges. This finding provided evidence that the cytoplasmic and nuclear functions of FMRP are uncoupled played by respective cytoplasmic and nuclear isoforms, potentially involving specific interactions. While interaction partners of cytoplasmic FMRP have been reported, the identity of nuclear FMRP isoform partners remains to be established. Using affinity purification coupled with mass spectrometry, we mapped the nuclear interactome of the FMRP isoform iso6 in U2OS. In doing so, we found FMRP nuclear interaction partners to be involved in RNA processing, pre-mRNA splicing, ribosome biogenesis, DNA replication and damage response, chromatin remodeling and chromosome segregation. By comparing interactions between nuclear iso6 and cytoplasmic iso1, we report a set of partners that bind specifically to the nuclear isoforms, mainly proteins involved in DNA-associated processes and proteasomal proteins, which is consistent with our finding that proteasome targets the nuclear FMRP iso6. The specific interactions with the nuclear isoform 6 are regulated by replication stress, while those with the cytoplasmic isoform 1 are largely insensitive to such stress, further supporting a specific role of nuclear isoforms in DNA damage response induced by replicative stress, potentially regulated by the proteasome.

Nuclear localization of STING1 competes with canonical signaling to activate AHR for commensal and intestinal homeostasis

Extensive studies demonstrate the importance of the STING1 (also known as STING) protein as a signaling hub that coordinates immune and autophagic responses to ectopic DNA in the cytoplasm. Here, we report a nuclear function of STING1 in driving the activation of the transcription factor aryl hydrocarbon receptor (AHR) to control gut microbiota composition and homeostasis. This function was independent of DNA sensing and autophagy and showed competitive inhibition with cytoplasmic cyclic guanosine monophosphate (GMP)-AMP synthase (CGAS)-STING1 signaling. Structurally, the cyclic dinucleotide binding domain of STING1 interacted with the AHR N-terminal domain. Proteomic analyses revealed that STING1-mediated transcriptional activation of AHR required additional nuclear partners, including positive and negative regulatory proteins. Although AHR ligands could rescue colitis pathology and dysbiosis in wild-type mice, this protection was abrogated by mutational inactivation of STING1. These findings establish a key framework for understanding the nuclear molecular crosstalk between the microbiota and the immune system.

Phosphorylation impacts GLE1 nuclear localization and association with DDX1

Gle1 regulates gene expression at multiple steps from transcription to mRNA export to translation under stressed and non-stressed conditions. To better understand Gle1 function in stressed human cells, specific antibodies were generated that recognized the phosphorylation of threonine residue 102 (T102) in Gle1. A series of in vitro kinase assays indicated that T102 phosphorylation serves as a priming event for further phosphorylation in Gle1's N-terminal low complexity cluster. Indirect immunofluorescence microscopy with the anti-Gle1-pT102 antibodies revealed that basally phosphorylated Gle1 was pre-dominantly nuclear with punctate distribution; however, under sodium arsenite-induced stress, more cytoplasmic localization was detected. Immunoprecipitation with the anti-Gle1-pT102 antibody resulted in co-isolation of Gle1-pT102 with the DEAD-box protein DDX1 in a phosphatase sensitive manner. This suggested Gle1 phosphorylation might be linked to its role in regulating DDX1 during transcription termination. Notably, whereas the total Gle1-DDX1 association was decreased when Gle1 nucleocytoplasmic shuttling was disrupted, co-isolation of Gle1-pT102 and DDX1 increased under the same conditions. Taken together, these studies demonstrated that Gle1 phosphorylation impacts its cellular distribution and potentially drives nuclear Gle1 functions in transcription termination. We propose a model wherein phosphorylation of Gle1 either reduces its nucleocytoplasmic shuttling capacity or increases its binding affinity with nuclear interaction partners.

In silico Analysis of SlNAC1 Transcription Factor, a Positive Modulator for Tomato Leaf Curl Virus

NAC proteins, part of the largest plant transcription factor family, play pivotal roles in plant development, and biotic and abiotic stresses. Among them, the SlNAC1 protein in tomato serves as a Negative regulator, enhancing tolerance to cold, bacterial, and viral diseases. However, our knowledge of SlNAC1 protein in silico analysis, evolutionary relationships, and protein interactions remains limited. We used in silico tools to study the SlNAC1 protein in tomato. Phylogenetic analysis showed it is closely related to other Solanaceae family members. Motif analysis revealed seven conserved motifs shared among Solanaceae members. The presence of NLS confirms its abundance in the nucleus. Protein-protein interaction analysis predicted ten potential nuclear partners, including five uncharacterized proteins and others involved in plant development. In silico expression, analysis showed abundant SlNAC1 presence in both fruit and roots. This study provides the role of SlNAC1 protein in tomato, shedding light on its evolutionary context and protein interactions.

National Security as a Value-Added Proposition for Advanced Nuclear Reactors: A U.S. Focus

The U.S. has transitioned from being the 20th-century global leader in civilian nuclear power to a nation searching for ways to revive its once-dominant nuclear enterprise. The future of U.S. civilian nuclear power transcends that of a science and technology issue and, fundamentally, is a policy issue. This is a policy paper that uses a nuclear power policy framework to analyze current and historical U.S. civilian nuclear power policy and to identify weaknesses and deficiencies that need to be overcome in order for the U.S. to (1) leverage advanced nuclear reactors as a domestic technology to meet energy security and reliability objectives under carbon constraints, (2) operationalize national security as a priority objective and (3) restore the U.S. as a major global exporter of nuclear technology. The results of this analysis indicate that the national security implications of U.S. nuclear power have been marginalized in general due to the domestic market challenges of competing with less expensive and oftentimes more socially acceptable technologies, as well as the international challenges of competing with state-owned nuclear enterprises. The results are then discussed and used for making three following policy recommendations: (1) conduct a U.S. nuclear industrial base review; (2) create a demand signal using U.S. military installations; and (3) shift away from a sell-side nuclear vendor model for global exports to a buy-side model brokered by a third-party integrator that can work with multiple U.S. nuclear partners.

원전의 인허가 규제 관련 쟁점과 과제

The nuclear power plant construction project faced a major crisis due to the Moon Jae-in government's nuclear power plant policy. Construction of Shin-Kori Units 5 and 6 was resumed as recommended by the Public Deliberation Committee, but plans to build Shin-Hanul Units 3 and 4 in Uljin and Cheonji Units 1 and 2 in Yeongdeok were nullified. As a result, residents of areas scheduled for nuclear power plant construction, such as Uljin and Yeongdeok, had to endure disadvantages due to economic downturn, job loss, and infringement of property rights. The Moon Jae-in government's policy of nuclear power plant was raised in the form of a presidential election pledge to join the energy conversion trend of some countries, including Germany. This was a big event that changed the basis of the national energy policy that had been maintained for a long time, and caused controversy over whether it was desirable or not in terms of industrial and energy security. Korea is a country with absolutely insufficient endowed energy resources, where it is importing more than 95% of its energy from abroad, and where it is difficult to connect to the power grid with foreign countries. If Korea gives up nuclear power, it will not be able to secure a stable energy supply. It can cause serious problems in terms of energy security. Permission for nuclear power plant construction is related to the Basic Plan for Electricity Demand and Supply(BPEDS). Establishing the BPEDS, K-Water gets intent documents from power producer, and reviews whether or not to permit generation, then, it is reflected in the BPEDS. In other words, when a new nuclear power plant construction is reflected to the BPEDS in the licensing proceedings for construction of nuclear power plants. K-water establishes a basic construction plan, conducts an environmental impact assessment, and then applies for approval of the power source development project execution plan in accordance with Article 5 of the Power Source Development Promotion Act, finally receives a construction permit of the nuclear power plant. Seoul Administrative Court dismissed the appel because BPEDS was not the object of the appeal under the administrative disposition in the Decision 2018Guhap53344 Decided January 10, 2020. In the case of the 8th BPEDS, it is reasonable to admit its disposability because it can affect specifically individual rights and obligations immediately by just the plan was established. In principle, the BPEDS is an internal guideline for administrative agencies to establish various plans for electricity business, and is applicable only to administrative agencies. However, the cancellation of construction and suspension of construction according to the 8th BPEDS can be seen directly and concretely to affect the rights and obligations of the people, such as nuclear power plant partners, nuclear equipment manufacturers and suppliers, nuclear radiation safety management service companies, and merchants in the vicinity of nuclear power plants. The point to resuming the construction of Shin Hanul Units 3 and 4 is the environmental impact assessment. Shin Hanul Units 3 and 4 received an environmental impact assessment in 2016, but the evaluation period ended in August 2021 as its construction was halted due to the Moon Jae-in government's policy of nuclear power plant. Therefore, it is legally appropriate to conduct the environmental impact assessment again. In principle, it is correct to proceed with the environmental assessment again from the beginning. However, in the case of Shin Hanul Units 3 and 4, all procedures were suspended due to the government's decision to suspend construction, so there attribute a fault to the government. In addition, environmental impact assessment is to prepare measures to avoid, eliminate or reduce harmful environmental impacts by investigating, predicting, and evaluating the impact of the project on the environment in advance.

Nuclear High Mobility Group A2 (HMGA2) Interactome Revealed by Biotin Proximity Labeling.

The non-histone chromatin binding protein High Mobility Group AT-hook protein 2 (HMGA2) has important functions in chromatin remodeling, and genome maintenance and protection. Expression of HMGA2 is highest in embryonic stem cells, declines during cell differentiation and cell aging, but it is re-expressed in some cancers, where high HMGA2 expression frequently coincides with a poor prognosis. The nuclear functions of HMGA2 cannot be explained by binding to chromatin alone but involve complex interactions with other proteins that are incompletely understood. The present study used biotin proximity labeling, followed by proteomic analysis, to identify the nuclear interaction partners of HMGA2. We tested two different biotin ligase HMGA2 constructs (BioID2 and miniTurbo) with similar results, and identified known and new HMGA2 interaction partners, with functionalities mainly in chromatin biology. These HMGA2 biotin ligase fusion constructs offer exciting new possibilities for interactome discovery research, enabling the monitoring of nuclear HMGA2 interactomes during drug treatments.

Medicinal Chemistry Strategies for the Development of Inhibitors Disrupting β-Catenin's Interactions with Its Nuclear Partners.

Dysregulation of the Wnt/β-catenin signaling pathway is strongly associated with various aspects of cancer, including tumor initiation, proliferation, and metastasis as well as antitumor immunity, and presents a promising opportunity for cancer therapy. Wnt/β-catenin signaling activation increases nuclear dephosphorylated β-catenin levels, resulting in β-catenin binding to TCF and additional cotranscription factors, such as BCL9, CBP, and p300. Therefore, directly disrupting β-catenin's interactions with these nuclear partners holds promise for the effective and selective suppression of the aberrant activation of Wnt/β-catenin signaling. Herein, we summarize recent advances in biochemical techniques and medicinal chemistry strategies used to identify potent peptide-based and small-molecule inhibitors that directly disrupt β-catenin's interactions with its nuclear binding partners. We discuss the challenges involved in developing drug-like inhibitors that target the interactions of β-catenin and its nuclear binding partner into therapeutic agents.

Interactions governing transcriptional activity of nuclear receptors.

The key players in transcriptional regulation are transcription factors (TFs), proteins that bind specific DNA sequences. Several mechanisms exist to turn TFs 'on' and 'off', including ligand binding which induces conformational changes within TFs, subsequently influencing multiple inter- and intramolecular interactions to drive transcriptional responses. Nuclear receptors are a specific family of ligand-regulated TFs whose activity relies on interactions with DNA, coregulator proteins and other receptors. These multidomain proteins also undergo interdomain interactions on multiple levels, further modulating transcriptional outputs. Cooperation between these distinct interactions is critical for appropriate transcription and remains an intense area of investigation. In this review, we report and summarize recent findings that continue to advance our mechanistic understanding of how interactions between nuclear receptors and diverse partners influence transcription.

Combining affinity purification and mass spectrometry to define the network of the nuclear proteins interacting with the N-terminal region of FMRP.

Fragile X-Syndrome (FXS) represents the most common inherited form of intellectual disability and the leading monogenic cause of Autism Spectrum Disorders. In most cases, this disease results from the absence of expression of the protein FMRP encoded by the FMR1 gene (Fragile X messenger ribonucleoprotein 1). FMRP is mainly defined as a cytoplasmic RNA-binding protein regulating the local translation of thousands of target mRNAs. Interestingly, FMRP is also able to shuttle between the nucleus and the cytoplasm. However, to date, its roles in the nucleus of mammalian neurons are just emerging. To broaden our insight into the contribution of nuclear FMRP in mammalian neuronal physiology, we identified here a nuclear interactome of the protein by combining subcellular fractionation of rat forebrains with pull‐ down affinity purification and mass spectrometry analysis. By this approach, we listed 55 candidate nuclear partners. This interactome includes known nuclear FMRP-binding proteins as Adar or Rbm14 as well as several novel candidates, notably Ddx41, Poldip3, or Hnrnpa3 that we further validated by target‐specific approaches. Through our approach, we identified factors involved in different steps of mRNA biogenesis, as transcription, splicing, editing or nuclear export, revealing a potential central regulatory function of FMRP in the biogenesis of its target mRNAs. Therefore, our work considerably enlarges the nuclear proteins interaction network of FMRP in mammalian neurons and lays the basis for exciting future mechanistic studies deepening the roles of nuclear FMRP in neuronal physiology and the etiology of the FXS.

Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer

BackgroundTargeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L.MethodsWe combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes.ResultsDot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine–protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells.ConclusionsIdentification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.

Identified potential pathogenic variants of BARD1 in 1449 Chinese high-risk breast cancer patients.

e22525 Background: BARD1 (BRCA1-associated ring domain 1), nuclear partner of BRCA1, has been recognized as a breast cancer predisposition gene. Recently, two large-scale population-based case–control studies confirmed that germline loss-of-function mutations in BARD1 were associated with a risk of breast cancer. Since the germline mutation frequency of BARD1 is much less than that of BRCA1/2, the distinct mutation spectrum of BARD1 is still obscure, especially in Chinese breast cancer patients. To verify the utility of BARD1 genetic testing in Chinese population, we assessed the mutational frequency and spectrum of BARD1 in a sequential series of Chinese high-risk breast cancer patients. Methods: A cohort of high-risk breast cancer patients (n = 1449) were collected in Zhejiang Cancer Hospital from 2008 through 2020, including 608 familial breast cancer (BC) cases, 173 bilateral BC cases, 222 triple negative BC cases, 436 early-onset BC cases (≤40 years) and 10 male BC cases. The complete coding sequence and intron–exon boundaries of BARD1 were screened by a 98-gene panel sequencing assay. The SIFT, Polyphen2 and MutationTaster prediction programs were used for analyzing the effect of the variants of unknown significance (VUS) on the BARD1 protein function. Results: A total of three BARD1 pathogenic mutations (c.1348_1349delinsCAT, c.70_71insGT and c.373G > T) were identified, which had been all reported, and accounting for 0.2% (3/1449) in Chinese high-risk breast cancer patients. The frequencies were 0.33% (2/608) and 0.12% (1/841) in familial and non-familial breast cancer patients, respectively. In total, 19 VUSs including 17 missenses, one inframe deletion and one inframe insertion of BARD1 were identified in this study. Based on in silico analysis, nine BARD1 missense variants (c.76A > G, c.1912G > A, c.1693C > T, c.2191C > T, c.127C > A, c.1601C > T, c.443G > A, c.233G > A and c.1972C > T) were classified as potentially damaging. However, the two newly variants c.420_422del and c.68_69insTCCGGGAACGAGCCTCGTTCC still remain VUS, and the other eight variants were classified as benign. Conclusions: Our data presented the germline mutations of BARD1 in a large-scale Chinese high-risk breast cancer population, which accounted for 0.2% in our cohort. Moreover nine potential pathogenic variants were found based on in silico analysis. Clinically, these data may be helpful in genetic counseling of breast cancer patients with BARD1 germline mutation. While the further research on the VUS are needed.

NIPA (Nuclear Interaction Partner of ALK) Is Crucial for Effective NPM-ALK Mediated Lymphomagenesis.

The NPM-ALK fusion kinase is expressed in 60% of systemic anaplastic large-cell lymphomas (ALCL). A Nuclear Interaction Partner of ALK (NIPA) was identified as a binding partner of NPM-ALK. To identify the precise role of NIPA for NPM-ALK-driven lymphomagenesis, we investigated various NPM-ALK+ cell lines and mouse models. Nipa deletion in primary mouse embryonic fibroblasts resulted in reduced transformation ability and colony formation upon NPM-ALK expression. Downregulating NIPA in murine NPM-ALK+ Ba/F3 and human ALCL cells decreased their proliferation ability and demonstrated synergistic effects of ALK inhibition and NIPA knockdown. Comprehensive in vivo analyses using short- and long-latency transplantation mouse models with NPM-ALK+ bone marrow (BM) revealed that Nipa deletion inhibited NPM-ALK-induced tumorigenesis with prolonged survival and reduced spleen colonies. To avoid off-target effects, we combined Nipa deletion and NPM-ALK expression exclusively in T cells using a lineage-restricted murine ALCL-like model resembling human disease: control mice died from neoplastic T-cell infiltration, whereas mice transplanted with Lck-CreTG/wtNipaflox/flox NPM-ALK+ BM showed significantly prolonged survival. Immunophenotypic analyses indicated a characteristic ALCL-like phenotype in all recipients but revealed fewer “stem-cell-like” features of Nipa-deficient lymphomas compared to controls. Our results identify NIPA as a crucial player in effective NPM-ALK-driven ALCL-like disease in clinically relevant murine and cell-based models.