Actin-dependent Regulator Of Chromatin Research Articles

SMARCA2 and SMARCA4 Participate in DNA Damage Repair.

The switching/sucrose non-fermentable (SWI/SNF) Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A (SMARCA) member 2 and member 4 (SMARCA2/4) are paralogs and act as the key enzymatic subunits in the SWI/SNF complex for chromatin remodeling. However, the role of SMARCA2/4 in DNA damage response remains unclear. Laser microirradiation assays were performed to examine the key domains of SMARCA2/4 for the relocation of the SWI/SNF complex to DNA lesions. To examine the key factors that mediate the recruitment of SMARCA2/4, the relocation of SMARCA2/4 to DNA lesions was examined in HeLa cells treated with inhibitors of Ataxia-telangiectasia-mutated (ATM), Ataxia telangiectasia and Rad3-related protein (ATR), CREB-binding protein (CBP) and its homologue p300 (p300/CBP), or Poly (ADP-ribose) polymerase (PARP) 1/2 as well as in H2AX-deficient HeLa cells. Moreover, by concomitantly suppressing SMARCA2/4 with the small molecule inhibitor FHD286 or Compound 14, the function of SMARCA2/4 in Radiation sensitive 51 (RAD51) foci formation and homologous recombination repair was examined. Finally, using a colony formation assay, the synergistic effect of PARP inhibitors and SMARCA2/4 inhibitors on the suppression of tumor cell growth was examined. We show that SMARCA2/4 relocate to DNA lesions in response to DNA damage, which requires their ATPase activities. Moreover, these ATPase activities are also required for the relocation of other subunits in the SWI/SNF complex to DNA lesions. Interestingly, the relocation of SMARCA2/4 is independent of γH2AX, ATM, ATR, p300/CBP, or PARP1/2, indicating that it may directly recognize DNA lesions as a DNA damage sensor. Lacking SMARCA2/4 prolongs the retention of γH2AX, Ring Finger Protein 8 (RNF8) and Breast cancer susceptibility gene 1 (BRCA1) at DNA lesions and impairs RAD51-dependent homologous recombination repair. Furthermore, the treatment of an SMARCA2/4 inhibitor sensitizes tumor cells to PARP inhibitor treatment. This study reveals SMARCA2/4 as a DNA damage repair factor for double-strand break repair.

ALTering Cancer by Triggering Telomere Replication Stress through the Stabilization of Promoter G-Quadruplex in SMARCAL1.

Most of the human cancers are dependent on telomerase to extend the telomeres. But ∼10% of all cancers use a telomerase-independent, homologous recombination mediated pathway called alternative lengthening of telomeres (ALT). Due to the poor prognosis, ALT status is not being considered yet in the diagnosis of cancer. No such specific treatment is available to date for ALT positive cancers. ALT positive cancers are dependent on replication stress to deploy DNA repair pathways to the telomeres to execute homologous recombination mediated telomere extension. SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1) is associated with the ALT telomeres to resolve replication stress thus providing telomere stability. Thus, the dependency on replication stress regulatory factors like SMARCAL1 made it a suitable therapeutic target for the treatment of ALT positive cancers. In this study, we found a significant downregulation of SMARCAL1 expression by stabilizing the G-quadruplex (G4) motif found in the promoter of SMARCAL1 by potent G4 stabilizers, like TMPyP4 and BRACO-19. SMARCAL1 downregulation led toward the increased localization of PML (promyelocytic leukemia) bodies in ALT telomeres and triggered the formation of APBs (ALT-associated promyelocytic leukemia bodies) in ALT positive cell lines, increasing telomere replication stress and DNA damage at a genomic level. Induction of replication stress and hyper-recombinogenic phenotype in ALT positive cells mediated by G4 stabilizing molecules already highlighted their possible application as a new therapeutic window to target ALT positive tumors. In accordance with this, our study will also provide a valuable insight toward the development of G4-based ALT therapeutics targeting SMARCAL1.

A Critical Review of the Impact of SMARCA4 Mutations on Survival Outcomes in Non-Small Cell Lung Cancer.

This critical review investigates the impact of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4) mutations on survival outcomes in non-small cell lung cancer (NSCLC) through an analysis of 21 peer-reviewed articles. Survival analyses across this review demonstrated consistently worse outcomes for SMARCA4-mutated vs. SMARCA4 wild-type NSCLC patients, specifically emphasizing class 1 truncating mutations as an independent factor for poor overall survival. In addition, this review explores the clinicopathologic characteristics of SMARCA4 mutations and their impact on various treatment modalities, including immune checkpoint inhibitors (ICIs) both with and without Kirsten rat sarcoma viral oncogene homolog (KRAS) co-mutations. The potential ineffectiveness of ICI treatment in NSCLC is explored through the impact of SMARCA4/KRAS co-mutations on the tumor microenvironment. Moreover, this NSCLC review consistently reported statistically worse overall survival outcomes for SMARCA4/KRAS co-mutations than SMARCA4 wild-type/KRAS-mutated cohorts, extending across ICIs, chemo-immunotherapy (CIT), and KRAS G12C inhibitors. Designing prospective clinical SMARCA4-mutated or SMARCA4/KRAS co-mutated NSCLC trials to evaluate targeted therapies and immunotherapy may lead to a better understanding of how to improve cancer patients' outcomes and survival rates.

Detection and Significance of Molecular Markers in Immunotherapy and Targeted Therapy of Colorectal Cancer in Tibet

Objective To study the expression of SWI/SNF-related,matrix-associated,actin-dependent regulator of chromatin,subfamily A,member 4(SMARCA4)/Brahma-related gene 1,V-raf murine sarcoma viral oncogene homolog B(BRAF),P53,programmed cell death protein-1(PD-1),and programmed death-ligand 1(PD-L1),and changes in the expression of BRAF and neurotrophic tyrosine receptor kinase(NTRK) in the patients with colorectal cancer in Tibet,thereby providing a basis for targeted therapy and immunotherapy for this disease in Tibet. Methods A total of 64 patients with colorectal cancer resected in the Tibet Autonomous Region People's Hospital from January 2015 to July 2021 were enrolled in this study.The expression of SMARCA4,BRAF,P53,PD-1,and PD-L1 was detected by immunohistochemical staining.The gene fusion involving NTRK1,NTRK2,and NTRK3 was detected by fluorescence in situ hybridization,and the BRAF V600E gene mutation by polymerase chain reaction. Results The 64 patients with colorectal cancer were at a male-to-female ratio of 1.21∶1,with the mean age of (56.59±13.27) years.The tumors were located in the colon in 46(71.88%) patients and in the rectum in 18(28.12%) patients.Sixty(93.75%) patients presented adenocarcinoma,and 4(6.25%) patients presented other types of tumors.The patients in T1/T2 and T3/T4 phases accounted for 17.19%(n=11) and 82.81%(n=53),respectively.Lymph node metastasis occurred in 24(37.50%) patients.The immunohistochemical staining results showed partially down-regulated or absent expression of SMARCA4 in 1(1.56%) patient,positive BRAF expression in 4(6.25%) patients,and mutant expression of P53 in 35(54.69%) patients.The PD-1-expressing tumor associated immune cell was proportion score<10% in 45(70.31%) patients and≥10% in 19(29.69%) patients.The PD-L1 combined positive score was<10 in 52(81.25%) patients and≥10 in 12(18.75%) patients.The gene fusion of NTRK1,NTRK2,and NTRK3 was negative in all the patients,and BRAF V600E gene mutation was positive in 4(6.25%) patients.The SMARCA4 gene alteration was not detected in the patient with partial expression missing of SMARCA4.The PD-L1 combine positive score was correlated with the deficient mismatch repair(dMMR)/microsatellite instability-high (MSI-H) and the PD-1 expression (χ2=10.223,P=0.001;χ2=11.979,P=0.001). Conclusions The down-regulated or absent SMARCA4 expression and NTRK gene fusion are rare in the patients with colorectal cancer in Tibet.A few patients present BRAF V600E gene mutations,and Pan-TRK and BRAF expression can be used for the primary screening of NTRK gene fusion and BRAF gene mutation.The patients with dMMR/MSI-H are prone to high expression of PD-L1 and expected to benefit from immunotherapy.No significant correlation exists between P53 mutation and PD-L1 expression.The high expression of PD-1 is positively correlated with the high expression of PD-L1.

Esophageal carcinoma with SMARCA4 mutation: a narrative review for this rare entity.

Esophageal carcinoma with switch/sucrose nonfermenting (SWI/SNF)-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4) mutation is a rare variant of malignant esophageal epithelial neoplasm, which is characterized by the loss of SMARCA4/BRG1 protein on immunohistochemistry or alterations in the SMARCA4 gene on sequencing. Only a few case series and case reports of esophageal carcinoma with SMARCA4 mutations have been published in the English literature; the rarity of the disease poses significant diagnostic challenges for surgical pathologists and could potentially lead to delayed or suboptimal patient care. Herein, we reviewed the available literature on esophageal carcinoma with SMARCA4 mutations to discuss its epidemiology, clinical presentation, pathological and molecular features, diagnostic challenges, treatment, and prognosis. The PubMed, Scopus, Ovid, and Google Scholar databases were extensively reviewed. The references included in the articles were cross-examined to identify any missing articles. We searched for all published literature on esophageal carcinoma with SMARCA4 mutations from inception of the databases to date. Esophageal carcinoma with SMARCA4 mutations is most common in middle-aged and older men. Barrett esophagus and gastroesophageal reflux disease (GERD) are the most associated risk factors. Dysphagia was the most common initial clinical presentation. Esophagogastroduodenoscopy (EGD) is the preferred diagnostic modality. Microscopically, the tumor cells exhibited epithelioid features mixed with variable components of rhabdoid and glandular differentiation. The tumor cells showed variable immunoreactivity for cytokeratin and sometimes weakly expressed neuroendocrine or B-lymphocyte markers (Pax5), which are potential diagnostic pitfalls. Melanoma marker tests showed negative results. The SMARCB1/INI1 protein remains intact, and a definitive diagnosis necessitates the presence of either SMARCA4/BRG1 protein loss or SMARCA4 gene mutations. Esophageal carcinoma with SMARCA4 mutations shows overly aggressive behavior and presents with advanced stages of disease; most patients succumb to the disease within 1 year of initial diagnosis. Esophageal carcinoma with SMARCA4 mutation is an overly aggressive disease, and further research on the affected molecular pathway may help improve its prognosis.

Recurrence/prognosis estimation using a molecularly positive surgical margin-based model calls for alternative curative strategies in pIIIA/N2 NSCLC.

Stage pIIIA/N2 non-small cell lung cancer (NSCLC) is primarily treated by complete surgical resection combined with neoadjuvant/adjuvant therapies. However, up to 40% of patients experience tumor recurrence. Here, we studied 119 stage pIIIA/N2 NSCLC patients who received complete surgery plus adjuvant chemotherapy (CT) or chemoradiotherapy (CRT). The paired tumor and resection margin samples were analyzed using next-generation sequencing (NGS). Although all patients were classified as negative resection margins by histologic methods, NGS revealed that 47.1% of them had molecularly positive surgical margins. Patients who tested positive for NGS-detected residual tumors had significantly shorter disease-free survival (DFS) (P = 0.002). Additionally, metastatic lymph node ratio, erb-b2 receptor tyrosine kinase 2 (ERBB2) mutations, and SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) mutations were also independently associated with DFS. We used these four features to construct a COX model that could effectively estimate recurrence risk and prognosis. Notably, mutational profiling through broad-panel NGS could more sensitively detect residual tumors than the conventional histologic methods. Adjuvant CT and adjuvant CRT exhibited no significant difference in eliminating locoregional recurrence risk for stage pIIIA/N2 NSCLC patients with molecularly positive surgical margins.

LncRNA UCA1 promotes tumor progression by targeting SMARCD3 in cervical cancer.

Long noncoding RNA urothelial carcinoma associated 1(UCA1) has been identified as a key molecule in human cancers. However, its functional implications remain unspecified in the context of cervical cancer (CC). This research aims to identify the regulatory mechanism of UCA1 in CC. UCA1 was identified through microarray and confirmed through a quantitative real-time polymerase chain reaction. Proteins that bind with UCA1 were recognized using RNA pull-down assays along with RNA immunoprecipitation. Ubiquitination assays and coimmunoprecipitation were performed to explore the molecular mechanisms of the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3 (SMARCD3) downregulated in CC. The effects of UCA1 and SMARCD3 on the progression of CC were investigated through gain- and loss-of-function assays and xenograft tumor formation in vivo. In this study, UCA1 was found to be upregulated in CC cells as well as in human plasma exosomes for the first time. Functional studies indicated that UCA1 promotes CC progression. Mechanically, UCA1 downregulated the SMARCD3 protein stabilization by promoting SMARCD3 ubiquitination. Taken together, we revealed that the UCA1/SMARCD3 axis promoted CC progression, which could provide a new therapeutic target for CC.

Loss of SMARCA4 Leads to an Impaired Hematopoiesis in Mice

SMARCA4 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a) is the central ATPase containing enzyme in the SWI/SNF- (switch/sucrose non-fermentable) complex, which regulates chromatin accessibility and thereby influences gene transcription. By acting as a transcriptional activator or repressor in different cell types, SMARCA4 and the SWI/SNF complex have various functions. Our aim was to understand the function of SMARCA4 in healthy hematopoiesis in order to then apply our findings to different hematologic diseases in a second step. To investigate the role of SMARCA4 in haematopoiesis and hematopoietic stem cells, we created a transgenic Smarca4 knockout mouse model. An inducible knockout system was chosen since early embryonic knockout of SMARCA4, which is crucially involved in embryonic development leads to a lethal phenotype. The Cre/loxP system was used to induce homozygous Smarca4 knockout under the control of the hematopoiesis-specific promotor Mx-1. We then performed blood counts, bone marrow smears and RNA sequencing, including analysis of RNA signature of the different cell types in the bone marrow to decipher the composition of the bone marrow of Smarca 4 deficient mice. Blood count analysis showed that Smarca4 knockout mice have significantly lower leukocyte, reticulocyte, and thrombocyte counts. In addition, bone marrow showed lower colony forming capacity in colony formation assays compared to healthy cre-negative control mice. Bone marrow smears displayed aplasia of all cell lines in Smarca4 knockout mice with additional signs of dysplasia as hypolobulatedmegakaryocytes or nuclear blasts. RNA-sequencing revealed 749 upregulated and 520 downregulated genes with a log fold change ≥ +/- 1 in the bone marrow of Smarca4 knockout mice compared to cre-negative control mice. Relevant over represented pathways according to REACTOME data base included genes of generation of second messenger molecules, cell surface interactions at the vascular wall, and immunoregulatory interactions between lymphoid and non-lymphoid cells. Through cell type deconvolution (CIBERSORTx) of the bulk microarray gene expression data we estimated abundances for different cell types. Results revealed a decrease in granulocyte and macrophage progenitors (statistically significant) as well as in eosinophil progenitor cells, and in erythroid pre-colony forming units. Interestingly, both common myeloid and common lymphoid progenitors were increased. It seems as if the progenitor cells accumulate and take a different path when Smarca4 is knocked out in these cells (figure 2). We conclude that SMARCA4 is crucial for cell growth and differentiation of progenitor cells in the bone marrow during hematopoiesis since a Smarca4 knockout leads to differentiation arrest and accumulation of distinct progenitor cells and pancytopenia in peripheral blood. Figure 1: Abundances for 23 cell populations in Smarca4 knockout (KO) and wildtype (WT) samples were estimated using CIBERSORTx. Bubble plot showing Log2 fold changes of average abundances in KO vs WT with accumulation of distinct progenitor cells in the bone marrow of Smarca4 deficient mice. As underlying signature-matrix the haemopedia gene set was used. Bubbles with black borders indicate significance of difference between samples.

Clinical and transcriptomic characteristics of a novel SMARCD2 mutation that disrupts neutrophil maturation and function.

We report a novel case of SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2) mutation successfully treated with hematopoietic stem cell transplantation. The female patient presented delayed cord separation, chronic diarrhea, skin abscesses, skeletal dysmorphisms, and neutropenia with specific granule deficiency. Analysis of the transcriptomic profile of peripheral blood sorted mature and immature SMARCD2 neutrophils showed defective maturation process that associated with altered expression of genes related to specific, azurophilic, and gelatinase granules, such as LTF, CRISP3, PTX3, and CHI3L1. These abnormalities account for the prevalence of immature neutrophils in the peripheral blood, impaired function, and deregulated inflammatory responses.

Regulation of 4-HNE via SMARCA4 Is Associated with Worse Clinical Outcomes in Hepatocellular Carcinoma.

Accumulation of 4-hydroxynonenal (4-HNE), a marker of lipid peroxidation, has various favorable and unfavorable effects on cancer cells; however, the clinicopathological significance of its accumulation in hepatocellular carcinoma (HCC) and its metabolic pathway remain unknown. This study analyzed 4-HNE accumulation and its clinicopathological significance in HCC. Of the 221 cases, 160 showed relatively low accumulation of 4-HNE in HCC tissues, which was an independent prognostic predictor. No correlation was found between 4-HNE accumulation and the expression of the antioxidant enzymes glutathione peroxidase 4, ferroptosis suppressor protein 1, and guanosine triphosphate cyclohydrolase 1. Therefore, we hypothesized that 4-HNE metabolism is up-regulated in HCC. A database search was focused on the transcriptional regulation of aldo-keto reductases, alcohol dehydrogenases, and glutathione-S-transferases, which are the metabolic enzymes of 4-HNE, and seven candidate transcription factor genes were selected. Among the candidate genes, the knockdown of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4) increased 4-HNE accumulation. Immunohistochemical analysis revealed an inverse correlation between 4-HNE accumulation and SMARCA4 expression. These results suggest that SMARCA4 regulates 4-HNE metabolism in HCC. Therefore, targeting SMARCA4 provides a basis for a new therapeutic strategy for HCC via 4-HNE accumulation and increased cytotoxicity.

Missense Mutation in Human CHD4 Causes Ventricular Noncompaction by Repressing ADAMTS1.

Left ventricular noncompaction (LVNC) is a prevalent cardiomyopathy associated with excessive trabeculation and thin compact myocardium. Patients with LVNC are vulnerable to cardiac dysfunction and at high risk of sudden death. Although sporadic and inherited mutations in cardiac genes are implicated in LVNC, understanding of the mechanisms responsible for human LVNC is limited. We screened the complete exome sequence database of the Pediatrics Cardiac Genomics Consortium and identified a cohort with a de novo CHD4 (chromodomain helicase DNA-binding protein 4) proband, CHD4M202I, with congenital heart defects. We engineered a humanized mouse model of CHD4M202I (mouse CHD4M195I). Histological analysis, immunohistochemistry, flow cytometry, transmission electron microscopy, and echocardiography were used to analyze cardiac anatomy and function. Ex vivo culture, immunopurification coupled with mass spectrometry, transcriptional profiling, and chromatin immunoprecipitation were performed to deduce the mechanism of CHD4M195I-mediated ventricular wall defects. CHD4M195I/M195I mice developed biventricular hypertrabeculation and noncompaction and died at birth. Proliferation of cardiomyocytes was significantly increased in CHD4M195I hearts, and the excessive trabeculation was associated with accumulation of ECM (extracellular matrix) proteins and a reduction of ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif 1), an ECM protease. We rescued the hyperproliferation and hypertrabeculation defects in CHD4M195I hearts by administration of ADAMTS1. Mechanistically, the CHD4M195I protein showed augmented affinity to endocardial BRG1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4). This enhanced affinity resulted in the failure of derepression of Adamts1 transcription such that ADAMTS1-mediated trabeculation termination was impaired. Our study reveals how a single mutation in the chromatin remodeler CHD4, in mice or humans, modulates ventricular chamber maturation and that cardiac defects associated with the missense mutation CHD4M195I can be attenuated by the administration of ADAMTS1.

Ovarian high-grade serous carcinoma cells with low SMARCA4 expression and high SMARCA2 expression contribute to platinum resistance.

Platinum resistance is a major obstacle to the treatment of ovarian cancer and is correlated with poor clinical outcomes. Intratumor heterogeneity plays a key role in chemoresistance. Recent studies have emphasized the contributions of genetic and epigenetic factors to the development of intratumor heterogeneity. Although the clinical significance of multi-subunit chromatin remodeler, switch/sucrose nonfermenting (SWI/SNF) complexes in cancers has been reported, the impacts of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4/subfamily A, member 2 (SMARCA4/A2) expression patterns in human cancer tissues have not been fully elucidated. Here, we show that low expression of SMARCA4 and high expression of SMARCA2 are associated with platinum resistance in ovarian high-grade serous carcinoma (HGSC) cells. We used fluorescence multiplex immunohistochemistry (fmIHC) to study resected specimens; we examined heterogeneity in human HGSC tissues at the single-cell level, which revealed that the proportion of cells with the SMARCA4low /SMARCA2high phenotype was positively correlated with clinical platinum-resistant recurrence. We used stable transfection of SMARCA2 and siRNA knockdown of SMARCA4 to generate HGSC cells with the SMARCA4low /SMARCA2high phenotype; these cells had the greatest resistance to carboplatin. Bioinformatics analyses revealed that the underlying mechanism involved in substantial alterations to chromatin accessibility and resultant fibroblast growth factor (FGF) signaling activation, MAPK pathway activation, BCL2 overexpression, and reduced carboplatin-induced apoptosis; these were confirmed by in vitro functional experiments. Furthermore, in vivo experiments in an animal model demonstrated that combination therapy with carboplatin and a fibroblast growth factor receptor (FGFR) inhibitor promoted cell death in HGSC xenografts. Taken together, these observations reveal a specific subpopulation of HGSC cells that is associated with clinical chemoresistance, which may lead to the establishment of a histopathological prediction system for carboplatin response. Our findings may facilitate the development of novel therapeutic strategies for platinum-resistant HGSC cells. © 2023 The Pathological Society of Great Britain and Ireland.

MiR-199a-5p-Regulated SMARCA4 Promotes Oral Squamous Cell Carcinoma Tumorigenesis.

SWI/SNF related, matrix associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4, also known as BRG1), an ATPase subunit of the switch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex, plays an important regulatory role in many cytogenetic and cytological processes during cancer development. However, the biological function and mechanism of SMARCA4 in oral squamous cell carcinoma (OSCC) remain unclear. The present study aimed to investigate the role of SMARCA4 in OSCC and its potential mechanism. Using a tissue microarray, SMARCA4 expression was found to be highly upregulated in OSCC tissues. In addition, SMARCA4 upregulate expression led to increased migration and invasion of OSCC cells in vitro, as well as tumor growth and invasion in vivo. These events were associated with the promotion of epithelial-mesenchymal transition (EMT). Bioinformatic analysis and luciferase reporter assay confirmed that SMARCA4 is a target gene of microRNA miR-199a-5p. Further mechanistic studies showed that the miR-199a-5p regulated SMARCA4 can promote the invasion and metastasis of tumor cells through EMT. These findings indicate that the miR-199a-5p- SMARCA4 axis plays a role in tumorigenesis by promoting OSCC cell invasion and metastasis through EMT regulation. Our findings provide insights into the role of SMARCA4 in OSCC and the mechanism involved, which may have important implications for therapeutic purposes.

Chromatin Remodeling Factor SMARCA5 is Essential for Hippocampal Memory Maintenance via Metabolic Pathways in Mice.

Gene transcription and new protein synthesis regulated by epigenetics play integral roles in the formation of new memories. However, as an important part of epigenetics, the function of chromatin remodeling in learning and memory has been less studied. Here, we showed that SMARCA5 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 5), a critical chromatin remodeler, was responsible for hippocampus-dependent memory maintenance and neurogenesis. Using proteomics analysis, we found protein expression changes in the hippocampal dentate gyrus (DG) after the knockdown of SMARCA5 during contextual fear conditioning (CFC) memory maintenance in mice. Moreover, SMARCA5 was revealed to participate in CFC memory maintenance via modulating the proteins of metabolic pathways such as nucleoside diphosphate kinase-3 (NME3) and aminoacylase 1 (ACY1). This work is the first to describe the role of SMARCA5 in memory maintenance and to demonstrate the involvement of metabolic pathways regulated by SMARCA5 in learning and memory.

Circular RNA SMARCA5 inhibits cholangiocarcinoma via microRNA-95-3p/tumor necrosis factor receptor associated factor 3 axis.

Enhancing research indicatedthat circular RNA (circRNA) acted a critical part in cholangiocarcinoma (CHOL) development. This research aims to discover the role of circRNA SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 5 (SMARCA5) in CHOL bio-progression, which has been proved to be downregulated in CHOL tissues. In this study, quantitative reverse transcription polymerase chain reaction was used to reveal the level and linkage of circRNA SMARCA5, miRNA-95-3p and TNF receptor-associated factor 3 gene (TRAF3) in CHOL tissues and cancer cells. The target sites of circRNA SMARCA5 and miRNA-95-3p were forecast by Starbase, and Targetscan was conducted to forecast the potential linkage points of TRAF3 and miRNA-95-3p, and which is affirmed by double luciferase reporter assay. CCK-8 and flow cytometry assay was carried to indicate cell viability. And apoptosis-related protein was counted by caspase3 activity and Western blot assay. CircRNA SMARCA5 was downregulated in CHOL cell lines and cancer samples. Besides, over-expression of SMARCA5 inhibited cell growth and promoted apoptotic rate. Dual-luciferase reporter assays presented that miRNA-95-3p could link with circRNA SMARCA5. Moreover, miRNA-95-3p was discovered highly expressed in CHOL. Interference of miRNA-95-3p repressed cell proliferation and raised the apoptosis. Importantly, TRAF3 was validated to be a downstream of miRNA-95-3p. Strengthen of miRNA-95-3p reversed the inhibitory impact of circRNA SMARCA5-plasmid transfection, and the results of miRNA-95-3p inhibitor were reversed by si-TRAF3. CircRNA SMARCA5 is involved in CHOL development by interosculating miRNA-95-3p/TRAF3 axis and may become a novel approach for treating CHOL.

An Update on WHO Classification of Thoracic Tumours 2021- Newly Described Entities and Terminologies

The 5th edition of the World Health Organisation (WHO) “Classification of Thoracic Tumours” replaces the previous edition from 2015, which was released in 2021. The new edition includes specific diagnostic criteria for each entity and lays a stronger emphasis on diagnostic molecular pathology. Immunohistochemistry (IHC) was heavily promoted in 2015, as a way to improve classification precision. The book places more attention on molecular pathology developments for all tumour types in 2021. Classification based on microscopic biopsy samples of the characteristics of lung cancer is an alternative to resection-based categorisation. A grading system for invasive non mucinous adenocarcinomas has been developed, using the percentage of distinctive histological patterns found inside each tumour. Lung adenocarcinoma is also predisposed by Tumour Spread Through Air Spaces (STAS). A basaloid variety of Squamous Cell Carcinoma (SCC) and lymphoepithelial carcinoma was added to the SCC group. The ciliated muconodular papillary tumour and thoracic SMARCA4-deficient undifferentiated carcinoma are recent inclusions. Therefore, the following new terms and entities have been added to the WHO classification of 2021: (1) Thoracic SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4 (SMARCA4)-deficient undifferentiated tumour; (2) Bronchiolar adenoma/ciliated muconodular papillary tumour; (3) Primary pulmonary hyalinising clear cell carcinoma of lung; (4) Lypmphoepithelial carcinoma; (5) STAS.

14 In vitro transformation of ovarian surface epithelium links a mutation in SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A, Member 4 to familial small carcinoma of the ovary hypercalcemic type

Using a unique population of cells derived from a familial carrier of an SCCOHT-associated SMARCA4 mutation (SMARCA4. c.3081+1G>T), we sought to determine if non-cancerous cells would spontaneously transform during in vitro culture, thereby providing a model system for studying the formation of SCCOHT. Ovarian surface epithelium (OSE) derived from a familial carrier of a SCCOHT-associated mutation in the SMARCA4 gene (P590, SMARCA4. c.3081+1G>T, prophylactic oophorectomy) were serially passaged in vitro, and interrogated for their ability to acquire characteristics of cellular transformation. Cells were grown for multiple passages to determine if cells would spontaneously immortalize. Loss of contact-dependent inhibition was interrogated with focus-forming assays of confluent monolayers. The ability of cells to escape anoikis and grow independently as spheroids was interrogated in attachment-free growth conditions. Senescenceassociated beta-galactosidase (SA-beta-gal), cellular necrosis, and cellular apoptosis was also measured. All experiments were conducted in parallel with an age-matched OSE population derived from a benign ovarian surgery, P590 cells that were passaged over 12 twelve times, representing at least 40 cellular doublings, continued to proliferate in culture. In contrast, non-mutant P583 cells failed to grow beyond 11–12 passages. P590 cells grown to confluency spontaneously formed foci of multicellular aggregates, while P583 cells did not. In attachment-free culture conditions, P590 cells readily formed spheroids from both early-passage and late-passage cells. In contrast, P583 formed small spheroids that failed to expand in culture. Surprisingly, both P590 and P583 cells displayed elevated SA-betagal staining, with neither cell populations showing elevated necrosis or apoptosis markers. OSE harboring a SMARCA4 mutation associated with familial SCCOHT display several phenotypes consistent with early stages of cellular transformation. In contrast, non-mutant cells failed to display signs of transformation. Our data suggest that OSE may represent a unique cell population for studying the early stages of SCCOHT formation, providing a useful tool for developing improved diagnostic and therapeutic approaches.

Management of a Composite Pheochromocytoma (Pheochromocytoma/Neuroblastoma) in Adult Patient Recurring After Several Years: A Complex Case Report.

Pheochromocytoma/neuroblastoma composite tumors are rare entities for which little is known. We report an atypical case of a 39-year-old man with secondary bone locations of a composite tumor, 7 years after resection of adrenal neuroblastoma, with constitutional alteration of SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 whose role is unknown. The diagnosis of a peripheral neuroblastic tumor in adulthood is difficult and even more so when it is a composite tumor. In the absence of a standard of care, management is varied and discussions about treatment modalities for these patients are complex.

Abstract 1457: NELFE regulates chromatin accessibility to affect MYC induced transcription

Abstract Liver cancer incidence rates have more than tripled in the last 40 years and is projected to increase more than 40% in the next decade. Although progress has been made, there is still limited therapeutic options. Due to the various etiological factors associated in HCC development and late stage in which it is diagnosed, the five-year survival rate is only 20%. Molecularly, more than 80% of HCC has an alteration in MYC signaling. Yet, MYC remains undruggable and studies over the recent years show that targeting proteins that regulate MYC is a promising approach in developing anti-cancer therapies. One such protein is the RNA binding protein (RBP) Negative Elongation Factor E (NELFE). We recently demonstrated for the first-time that NELFE recruits MYC via protein-protein interactions to the chromatin to control the expression of pro-survival genes. Consistent with the role of NELFE as a regulator of MYC in these cancers, we showed that patients with aberrant NELFE expression concurrently have active MYC signaling and poor outcome. These studies suggest that the NELFE/MYC interplay may be important for HCC maintenance. Thus, our hypothesis is that NELFE modulates the chromatin landscape to facilitate MYC-induced transcription to promote HCC. To investigate NELFE’s effect on MYC-induced transcription, we performed NELFE and MYC ChIP-sequencing and ATAC-sequencing analyses on CRISPR/Cas9-mediated NELFE knockout HCC cells. We found NELFE depletion resulted in significant reduction in nucleosomal accessibility and MYC binding to the chromatin near promoter-proximal regions. Although NELFE depletion alters the chromatin landscape, we found it rarely interacts with the chromatin, specifically within MYC bound regions, suggesting that the MYC/NELFE interaction is facilitated by a third protein. NELFE co-immunoprecipitation followed by mass spectrometry analyses identified the protein SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily B, Member 1 (SMARCB1) as a potential MYC cofactor that facilitates the NELFE/MYC interaction. Immunoblotting analyses show NELFE interacts with SMARCB1 and MYC and loss of NELFE significantly reduced SMARCB1/MYC interactions, indicating that NELFE facilitates chromatin accessibility through SMARCB1, which is required for MYC-induced transcription. Future studies include investigating SMARCB1’s oncogenic role in HCC through NELFE/MYC signaling. Citation Format: Kai Zhang, Anna Barry, Ryan Lamm, Hien Dang. NELFE regulates chromatin accessibility to affect MYC induced transcription [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1457.

Generation of a mouse model of the neurodevelopmental disorder with dysmorphic facies and distal limb anomalies syndrome.

Heterozygous variants in bromodomain and plant homeodomain containing transcription factor (BPTF) cause the neurodevelopmental disorder with dysmorphic facies and distal limb anomalies (NEDDFL) syndrome (MIM#617755) characterized by intellectual disability, speech delay and postnatal microcephaly. BPTF functions within nucleosome and remodeling factor (NURF), a complex comprising sucrose non-fermenting like (SNF2L), an Imitation SWItching (ISWI) chromatin remodeling protein encoded by the SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 1 (SMARCA1) gene. Surprisingly, ablation of Smarca1 resulted in mice with enlarged brains, a direct contrast to the phenotype of NEDDFL patients. To model the NEDDFL syndrome, we generated forebrain-specific Bptf knockout (Bptf conditional Knockout (cKO)) mice. The Bptf cKO mice were born in normal Mendelian ratios, survived to adulthood but were smaller in size with severe cortical hypoplasia. Prolonged progenitor cell cycle length and a high incidence of cell death reduced the neuronal output. Cortical lamination was also disrupted with reduced proportions of deep layer neurons, and neuronal maturation defects that impaired the acquisition of distinct cell fates (e.g. COUP-TF-interacting protein 2 (Ctip2)+ neurons). RNAseq and pathway analysis identified altered expression of fate-determining transcription factors and the biological pathways involved in neural development, apoptotic signaling and amino acid biosynthesis. Dysregulated genes were enriched for MYC Proto-Oncogene, BHLH Transcription Factor (Myc)-binding sites, a known BPTF transcriptional cofactor. We propose the Bptf cKO mouse as a valuable model for further study of the NEDDFL syndrome.