Inhibition Of Gene Transcription Research Articles

Therapeutic Potential of Decoys for Prostate Cancers: A Review of Recent Updates.

Prostate cancer is ranked second among the most common male cancers. Androgen deprivation therapy (ADT) has long been the first-line treatment and the basis for all other therapies, reducing circulating androgens to castration levels and preventing disease development. Nevertheless, ADT monotherapy may not always limit disease development, and even at low testosterone levels, hormone-sensitive prostate cancer will become castration-resistant. Recent research demonstrates that prostate cancer can have a range of potentially actionable genetic abnormalities; no medications that target these variations have yet been shown to elicit therapeutic advantages. Despite their established efficacy in the management of other cancers, advanced genetic or immunological approaches are not regularly used to treat prostate cancer patients. As a result, there is an unmet demand for medicines that offer a better chance of survival than the existing castration- resistance prostate cancer (CRPC) therapy regimens. The use of oligodeoxynucleotides (ODN) and peptides in decoy technology have been developed as novel therapeutic approaches. Decoy ODNs bind to a particular transcription factor with high affinity and may suppress gene transcription. Peptide decoys bind to specific ligands with high specificity and inhibit signaling pathways. Recent evidence supports the notion that these techniques are promising and attractive in the fight against cancer. In the present review, we discuss the use of decoy technology as a novel therapeutic approach against prostate cancer.

Cellular remodeling and JAK inhibition promote zygotic gene expression in the Ciona germline

Transcription control is a major determinant of cell fate decisions in somatic tissues. By contrast, early germline fate specification in numerous vertebrate and invertebrate species relies extensively on RNA-level regulation, exerted on asymmetrically inherited maternal supplies, with little-to-no zygotic transcription. However delayed, a maternal-to-zygotic transition is nevertheless poised to complete the deployment of pre-gametic programs in the germline. Here, we focus on early germline specification in the tunicate Ciona to study zygotic genome activation. We first demonstrate that a peculiar cellular remodeling event excludes localized postplasmic Pem-1 mRNA, which encodes the general inhibitor of transcription. Subsequently, zygotic transcription begins in Pem-1-negative primordial germ cells (PGCs), as revealed by histochemical detection of elongating RNA Polymerase II, and nascent Mef2 transcripts. In addition, we uncover a provisional antagonism between JAK and MEK/BMPRI/GSK3 signaling, which controls the onset of zygotic gene expression, following cellular remodeling of PGCs. We propose a 2-step model for the onset of zygotic transcription in the Ciona germline and discuss the significance of germ plasm dislocation and remodeling in the context of developmental fate specification.

EH domain-containing protein 2 (EHD2): Overview, biological function, and therapeutic potential.

EH domain-containing protein 2 (EHD2) is a member of the EHD protein family and is mainly located in the plasma membrane, but can also be found in the cytoplasm and endosomes. EHD2 is also a nuclear-cytoplasmic shuttle protein. After entering the cell nuclear, EHD2 acts as a corepressor of transcription to inhibit gene transcription. EHD2 regulates a series of biological processes. As a key regulator of endocytic transport, EHD2 is involved in the formation and maintenance of endosomal tubules and vesicles, which are critical for the intracellular transport of proteins and other substances. The N-terminal of EHD2 is attached to the cell membrane, while its C-terminal binds to the actin-binding protein. After binding, EHD2 connects with the actin cytoskeleton, forming the curvature of the membrane and promoting cell endocytosis. EHD2 is also associated with membrane protein trafficking and receptor signaling, as well as in glucose metabolism and lipid metabolism. In this review, we highlight the recent advances in the function of EHD2 in various cellular processes and its potential implications in human diseases such as cancer and metabolic disease. We also discussed the prospects for the future of EHD2. EHD2 has a broad prospect as a therapeutic target for a variety of diseases. Further research is needed to explore its mechanism, which could pave the way for the development of targeted treatments.

Abstract 4579: DNMT1 determines the sensitivity of Class I HDAC inhibitor Psammaplin A to induce apoptosis and DNA damage in hematological malignant cells

Abstract Histone deacetylases (HDACs) and DNA methyltransferases (DNMTs) are overexpressed in cancer cells associated with dysregulated gene expression and tumor progression. DNMT1 partnered with Class I HDAC as complex to inhibit gene transcription. Several pan-HDAC inhibitors have been approved for T-cell lymphoma with evident side effects. More selective HDAC inhibitors need to be developed. Psammaplin A (PsA), a symmetrical bromotyrosine-derived disulfide dimer isolated from the Psammaplysilla sponge, is a selective Class I HDAC inhibitor. We tested its cell growth inhibition and apoptosis induction in 12 hematological malignant cell lines with 8 lines sensitive (more than 50% growth inhibition at 1 μM) and 4 lines insensitive (less than 20% growth inhibition at 1 μM). PsA induces acetylation of H3, but not α-tubulin, supporting the Class I HDAC inhibition selection. In the sensitive cell lines, PsA induces apoptosis with down-regulation of c-Flip and upregulation of NOXA as well as DNA damage detected by γ-H2A.X. RNA sequence analysis results confirm the regulation of PsA on NOXA, c-Flip and p21 gene expression. The four insensitive cell lines express high levels of DNMT1 with resistance to apoptosis induction. Silencing DNMT1 with siRNA sensitizes PsA-induced apoptosis with induction of NOXA and γ-H2A.X. Azacytidine, the DNMT1 degrader, in combination with PsA induce synergistic cell growth inhibition and apoptosis in vitro. PsA (20 mg/kg) plus Azacytidine (2.5 mg/kg) synergistically inhibit tumor growth in insensitive mantle cell lymphoma Jeko-1 xenografts, without detected toxic signs in organ pathological analysis. We find a new mechanism of DNMT1 controlling the sensitivity of Class I HDAC inhibitor PsA and provide a rationale of PsA with azacytidine for combination therapy. Citation Format: Yu Bao, Dan Liu, Zi Wu, Yumei He, Ping Gong, Samuel Waxman, Yongkui Jing. DNMT1 determines the sensitivity of Class I HDAC inhibitor Psammaplin A to induce apoptosis and DNA damage in hematological malignant cells [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 4579.

Abstract 6055: COOTO-Me (10e) acts as a molecular glue to target NCOR/HDAC3 degradation

Abstract Histone deacetylase 3 (HDAC3) is overexpressed in cancer cells and functions as transcription repression complex with nuclear receptor corepressor (NCOR) to inhibit gene transcription. The pan-HDAC inhibitors have been approved for T-cell lymphoma with evident side effects. The selective HDAC3 inhibitors needs to be developed. Proteolysis-targeting chimera (PROTAC) approach is taken to design HDAC3 degraders with unsolved bioavailability obstacle. We screened small molecules with HDAC3 degradation ability and found two small molecules derived from glycyrrhetinic acid, methyl 2-cyano-3-oxo-18β-olean-1,9(11), 12-trien-30-oate (COOTO, 10e) and methyl 2-cyano-3,12-dioxo-18β-olean-1,9(11)-dien-30-oate (CDODO-Me, 10d), selectively decreased HDAC3 protein with increased acetylation of Histone 3. We explored the mechanism of HDAC3 degradation and found that 10e interrupted the interaction of HDAC3 with NCOR and induced degradation of both HDAC3 and NCOR protein. Biotin labeled 10e shows binding to both HDAC3 and NCOR directly as well as to E3 ligases SIAH2 and ITCH. Mass spectrometry analysis profiling reveals covalent interactions of 10e with multiple cysteine sites of SIAH2 in the binding regions for NCOR and HDAC3. Silencing SIAH2 blocks degradation of HDAC3. N-Acetylcysteine and GSH block the binding of 10e to HDAC3, NCOR and SIAH2. In addition, 10e decreased the levels of c-FLIP protein and increased the levels of NOXA protein leading to apoptosis induction. These data suggest that 10e functions as a molecular glue to attract SIAH2 to HDAC3 and NCOR to cause degradation through covalent binding. Citation Format: Ping Gong, Min Huang, Xin Li, Haoling Wang, Samuel Waxman, Linxiang Zhao, Yongkui Jing. COOTO-Me (10e) acts as a molecular glue to target NCOR/HDAC3 degradation [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 6055.

HDAC7: a promising target in cancer.

Histones have a vital function as components of nucleosomes, which serve as the fundamental building blocks of chromatin. Histone deacetylases (HDACs), which target histones, suppress gene transcription by compacting chromatin. This implies that HDACs have a strong connection to the suppression of gene transcription. Histone deacetylase 7 (HDAC7), a member of the histone deacetylase family, may participate in multiple cellular pathophysiological processes and activate relevant signaling pathways to facilitate the progression of different tumors by exerting deacetylation. In recent years, HDAC7 has been increasingly studied in the pathogenesis of tumors. Studies that are pertinent have indicated that it has a significant impact on the growth and metastasis of tumors, the formation of the vascular microenvironment, and the emergence of resistance to drugs. Therefore, HDAC7 could potentially function as a potent predictor for tumor prognosis and a promising target for mitigating drug resistance in tumors. This review primarily concentrates on elucidating the structure and function of HDAC7, its involvement in the development of various tumors, and its interplay with relevant signaling pathways. Meanwhile, we briefly discuss the research direction and prospect of HDAC7.

Filamin A is involved in human intrahepatic cholangiocarcinoma aggressiveness and progression.

Intrahepatic cholangiocarcinoma (iCCA) is a primary liver tumour, characterized by poor prognosis and lack of effective therapy. The cytoskeleton protein Filamin A (FLNA) is involved in cancer progression and metastasis, including primary liver cancer. FLNA is cleaved by calpain, producing a 90 kDa fragment (FLNACT ) that can translocate to the nucleus and inhibit gene transcription. We herein aim to define the role of FLNA and its cleavage in iCCA carcinogenesis. We evaluated the expression and localization of FLNA and FLNACT in liver samples from iCCA patients (n = 82) revealing that FLNA expression was independently correlated with disease-free survival. Primary tumour cells isolated from resected iCCA patients expressed both FLNA and FLNACT , and bulk RNA sequencing revealed a significant enrichment of cell proliferation and cell motility pathways in iCCAs with high FLNA expression. Further, we defined the impact of FLNA and FLNACT on the proliferation and migration of primary iCCA cells (n = 3) and HuCCT1 cell line using silencing and Calpeptin, a calpain inhibitor. We observed that FLNA silencing decreased cell proliferation and migration and Calpeptin was able to reduce FLNACT expression in both the HuCCT1 and iCCA cells (p < .05 vs. control). Moreover, Calpeptin 100 μM decreased HuCCT1 and primary iCCA cell proliferation (p <.00001 vs. control) and migration (p < .05 vs. control). These findings demonstrate that FLNA is involved in human iCCA progression and calpeptin strongly decreased FLNACT expression, reducing cell proliferation and migration.

Histone deacetylase 9 exacerbates podocyte injury in hyperhomocysteinemia through epigenetic repression of Klotho

Although hyperhomocysteinemia (hHcys) has been recognized as an important independent risk factor in the progression of end-stage renal disease and the development of cardiovascular complications related to end-stage renal disease, the mechanisms triggering pathogenic actions of hHcys are not fully understood. The present study was mainly designed to investigate the role of HDACs in renal injury induced by hHcys. Firstly, we identified the expression patterns of HDACs and found that, among zinc-dependent HDACs, HDAC9 was preferentially upregulated in the kidney from mice with hHcys. Deficiency or pharmacological inhibition of HDAC9 ameliorated renal injury in mice with hHcys. Moreover, podocyte-specific deletion of HDAC9 significantly attenuated podocyte injury and proteinuria. In vitro, gene silencing of HDAC9 attenuated podocyte injury by inhibiting apoptosis, reducing oxidative stress and maintaining the expressions of podocyte slit diaphragm proteins. Mechanically, we proved for the first time that HDAC9 reduced the acetylation level of H3K9 in the promoter of Klotho, then inhibited gene transcription of Klotho, finally aggravating podocyte injury in hHcys. In conclusion, our results indicated that targeting of HDAC9 might be an attractive therapeutic strategy for the treatment of renal injury induced by hHcys.

Biomolecular insights into the inhibition of heavy metals on reductive dechlorination of 2,4,6-trichlorophenol in Pseudomonas sp. CP-1

Influences of heavy metal exposure to the organohalide respiration process and the related molecular mechanism remain poorly understood. In this study, a non-obligate organohalide respiring bacterium, Pseudomonas sp. strain CP-1, was isolated and its molecular response to the five types of commonly existed heavy metal ions were thoroughly investigated. All types of heavy metal ions posed inhibitory effects on 2,4,6-trichlorophenol dechlorination activity and cell growth with the varied degree. Exposure to Cu (II) showed the most serious inhibitive effects on dechlorination even at the lowest concentration of 0.05 mg/L, while the inhibition by As (V) was the least with the removal kinetic constant k decreased to 0.05 under 50 mg/L. Further, multi-omics analysis found compared with Cu (II), As (V) exposure led to the insignificant downregulation of a variety of biosynthesis processes, which would be one possible account for the less inhibited activity. More importantly, the inhibited mechanisms on the organohalide respiration catabolism of strain CP-1 were firstly revealed. Cu (II) stress severely downregulated NADH generation during TCA cycle and electron donation of organohalide respiration process, which might decrease the reducing power required for organohalide respiration. While both Cu (II) and As (Ⅴ) inhibited substrate level phosphorylation during TCA cycle, as well as electron transfer and ATP generation during organohalide respiration. Meanwhile, CprA-2 was confirmed as the responsible reductive dehalogenase in charge of 2,4,6-TCP dechlorination, and transcriptional and proteomic studies confirmed the directly inhibited gene transcription and expression of CprA-2. The in-depth reveal of inhibitory effects and mechanism gave theoretical supports for alleviating heavy metal inhibition on organohalide respiration activity in groundwater co-contaminated with organohalides and heavy metals.

Discovery of three-dimensional bicyclo[3.3.1]nonanols as novel heat shock protein 90 inhibitors

We developed an efficient synthetic method for constructing bicyclo[3.3.1]nonane, an sp3-carbon-rich three-dimensional scaffold consisting of two fused six-membered rings. Among the bicyclo[3.3.1]nonanols synthesized, several bicyclo[3.3.1]nonane derivatives were found to inhibit gene transcription by hypoxia-inducible factor-1 (HIF-1). The structure-activity relationship study revealed that the number of hydrophobic functional groups and a carboxylic acid moiety in the bicyclo[3.3.1]nonanols are important for inhibitory activities of both gene transcription by HIF-1 and cell growth. Bicyclo[3.3.1]nonanols fluctuated the amounts of client proteins of heat shock protein (HSP) 90 without inducing a heat shock response in cells and specifically inhibited the ATPase activity of HSP90. These results indicate that bicyclo[3.3.1]nonanols are novel HSP90 inhibitors with a different mechanism of action from conventional HSP90 inhibitors.

Carcinogenesis promotion in oral squamous cell carcinoma: KDM4A complex-mediated gene transcriptional suppression by LEF1.

Oral squamous cell carcinoma (OSCC) is the most prevalent cancer of the mouth, characterised by rapid progression and poor prognosis. Hence, an urgent need exists for the development of predictive targets for early diagnosis, prognosis determination, and clinical therapy. Dysregulation of lymphoid enhancer-binding factor 1 (LEF1), an important transcription factor involved in the Wnt-β-catenin pathway, contributes to the poor prognosis of OSCC. Herein, we aimed to explore the correlation between LEF1 and histone lysine demethylase 4 A (KDM4A). Results show that the KDM4A complex is recruited by LEF1 and specifically binds the LATS2 promoter region, thereby inhibiting its expression, and consequently promoting cell proliferation and impeding apoptosis in OSCC. We also established NOD/SCID mouse xenograft models using CAL-27 cells to conduct an in vivo analysis of the roles of LEF1 and KDM4A in tumour growth, and our findings show that cells stably suppressing LEF1 or KDM4A have markedly decreased tumour-initiating capacity. Overall, the results of this study demonstrate that LEF1 plays a pivotal role in OSCC development and has potential to serve as a target for early diagnosis and treatment of OSCC.

The Significant Role of microRNAs in Gliomas Angiogenesis: A Particular Focus on Molecular Mechanisms and Opportunities for Clinical Application.

MicroRNAs (miRNAs) are non-coding RNAs with only 20-22 nucleic acids that inhibit gene transcription and translation by binding to mRNA. MiRNAs have a diverse set of target genes and can alter most physiological processes, including cell cycle checkpoints, cell survival, and cell death mechanisms, affecting the growth, development, and invasion of various cancers, including gliomas. So optimum management of miRNA expression is essential for preserving a normal biological environment. Due to their small size, stability, and capability of specifically targeting oncogenes, miRNAs have emerged as a promising marker and new biopharmaceutical targeted therapy for glioma patients. This review focuses on the most common miRNAs associated with gliomagenesis and development by controlling glioma-determining markers such as angiogenesis. We also summarized the recent research about miRNA effects on signaling pathways, their mechanistic role and cellular targets in the development of gliomas angiogenesis. Strategies for miRNA-based therapeutic targets, as well as limitations in clinical applications, are also discussed.

ChREBP-β/TXNIP aggravates frucose-induced renal injury through triggering ferroptosis of renal tubular epithelial cells.

High fructose intake is an essential risk factor for kidney injury. However, the specific mechanism underlying high fructose-induced kidney injury remains unclarified. Carbohydrate response element-binding protein (ChREBP) is a key transcriptional activator that regulates fructose metabolism. ChREBP-β exhibits sustained activity due to the lack of a low glucose inhibitory domain, and is thus described as the active form of ChREBP. In this study, a mouse model with specific overexpression of ChREBP-β in the renal tubule was established by using the Cre/LoxP method. Quantitative proteomic analysis and experimental verification results suggest that ChREP-β overexpression leads to ferroptosis of renal tubular epithelial cells and kidney injury. ChREPB-β promotes the gene transcription of thioredoxin-interacting protein (TXNIP) and thereby increases its expression level. TXNIP is associated with activation of ferroptosis. TXNIP can initiate ferroptosis and eventually contribute to high fructose-induced renal tubular epithelial cell damage. Through down-regulating ChREBP-β, metformin can inhibit gene transcription of TXNIP, attenuate high fructose-induced ferroptosis in renal tubular epithelial cells, and alleviate kidney injury. In conclusion, ChREBP-β mediates fructose-induced ferroptosis of renal tubular epithelial cells, and metformin with a ChREBP-β inhibitory effect may be a potential treatment for ferroptosis of renal tubular epithelial cells.

G9a promotes immune suppression by targeting the Fbxw7/Notch pathway in glioma stem cells.

Immunotherapy for glioblastoma multiforme (GBM) is limited because of a strongly immunosuppressive tumor microenvironment (TME). Remodeling the immune TME is an effective strategy to eliminate GBM immunotherapy resistance. Glioma stem cells (GSCs) are inherently resistant to chemotherapy and radiotherapy and involved in immune evasion mechanism. This study aimed to investigate the effects of histone methyltransferases 2 (EHMT2 or G9a) on immunosuppressive TME and whether this effect was related to changes on cell stemness. Tumor-infiltrating immune cells were analyzed by flow cytometry and immunohistochemistry in orthotopic implanted glioma mice model. The gene expressions were measured by RT-qPCR, western blot, immunofluorescence, and flow cytometry. Cell viability was detected by CCK-8, and cell apoptosis and cytotoxicity were detected by flow cytometry. The interaction of G9a and F-box and WD repeat domain containing 7 (Fbxw7) promotor was verified by dual-luciferase reporter assay and chromatin immunoprecipitation. Downregulation of G9a retarded tumor growth and extended survival in an immunocompetent glioma mouse model, promoted the filtration of IFN-γ + CD4+ and CD8+ T lymphocytes, and suppressed the filtration of PD-1+ CD4+ and CD8+ T lymphocytes, myeloid-derived suppressor cells (MDSCs) and M2-like macrophages in TME. G9a inhibition decreased PD-L1 and increased MHC-I expressions by inactivating Notch pathway companying stemness decrease in GSCs. Mechanistically, G9a bound to Fbxw7, a Notch suppressor, to inhibit gene transcription through H3K9me2 of Fbxw7 promotor. G9a promotes stemness characteristics through binding Fbxw7 promotor to inhibit Fbxw7 transcription in GSCs, forming an immunosuppressive TME, which provides novel treatment strategies for targeting GSCs in antitumor immunotherapy.

Parishin A-loaded mesoporous silica nanoparticles modulate macrophage polarization to attenuate tendinopathy

Macrophages are involved mainly in the balance between inflammation and tenogenesis during the healing process of tendinopathy. However, etiological therapeutic strategies to efficiently treat tendinopathy by modulating macrophage state are still lacking. In this study, we find that a small molecule compound Parishin-A (PA) isolated from Gastrodia elata could promote anti-inflammatory M2 macrophage polarization by inhibiting gene transcription and protein phosphorylation of signal transducers and activators of transcription 1. Local injection or sustained delivery of PA by mesoporous silica nanoparticles (MSNs) could almost recover the native tendon’s dense parallel-aligned collagen matrix in collagenase-induced tendinopathy by modulating macrophage-mediated immune microenvironment and preventing heterotopic ossification. Especially, MSNs decrease doses of PA, frequency of injection and yield preferable therapeutic effects. Mechanistically, intervention with PA could indirectly inhibit activation of mammalian target of rapamycin to repress chondrogenic and osteogenic differentiation of tendon stem/progenitor cells by influencing macrophage inflammatory cytokine secretion. Together, pharmacological intervention with a natural small-molecule compound to modulate macrophage status appears to be a promising strategy for tendinopathy treatment.

EZH2 W113C is a gain-of-function mutation in B-cell lymphoma enabling both PRC2 methyltransferase activation and tazemetostat resistance

Polycomb Repressive Complex2 (PRC2) suppresses gene transcription by methylating lysine 27 of histone H3 (H3K27) and plays critical roles in embryonic development. Among the core PRC2 subunits, EZH2 is the catalytic subunit and EED allosterically activates EZH2 upon binding trimethylated H3K27 (H3K27me3). Activating mutations on Y641, A677, and A687 within the enzymatic SET domain of EZH2 have been associated with enhanced H3K27me3 and tumorigenicity of many cancers including B-cell lymphoma and melanoma. To tackle the critical residues outside the EZH2 SET domain, we examined EZH2 mutations in lymphoma from cancer genome databases and identified a novel gain-of-function (GoF) mutation W113C, which increases H3K27me3 in vitro and in vivo and promotes CDKN2A silencing to a similar level as the EZH2 Y641F. Different from other GoF mutations, this mutation is located in the SAL motif at the EZH2 N-terminus, which stabilizes the SET domain and facilitates substrate binding. This may explain how the W113C mutation increases PRC2 activity. Tazemetostat is an FDA-approved EZH2 binding inhibitor for follicular lymphoma treatment. Intriguingly, the W113C mutation leads to tazemetostat-resistance in cell and in vivo in both H3K27 methylation and tumor proliferation. Another class of allosteric PRC2 inhibitor binding EED overcomes the resistance, effectively decreases H3K27me3, and blocks tumor proliferation in cells expressing EZH2 W113C. As this mutation is originally identified from lymphoma samples, our results demonstrated its activating characteristic and the deleterious consequence, provide insights on PRC2 regulation, and support the continued exploration of treatment optimization for lymphoma patients.

Investigation of the Interaction between Aloe vera Anthraquinone Metabolites and c-Myc and C-Kit G-Quadruplex DNA Structures

G-quadruplexes are nucleotide sequences present in the promoter region of numerous oncogenes, having a key role in the suppression of gene transcription. Recently, the binding of anthraquinones from Aloe vera to G-quadruplex structures has been studied through various physico-chemical techniques. Intrigued by the reported results, we investigated the affinity of aloe emodin, aloe emodin-8-glucoside, and aloin to selected G-quadruplex nucleotide sequences by NMR spectroscopy. The structural determinants for the formation of the ligand/nucleotide complexes were elucidated and a model of the interactions between the tested compounds and C-Kit and c-Myc G-quadruplex DNA structures was built by integrated NMR and molecular modeling studies. Overall, the obtained results confirmed and implemented the previously reported findings, pointing out the complementarity of the different approaches and their contribution to a more detailed overview of the ligand/nucleotide complex formation. Furthermore, the proposed models of interaction could pave the way to the design of new nature-derived compounds endowed with increased G-quadruplex stabilizing activity.

Sex Dimorphism in the Bone Marrow Niche Contributes to Gender Difference in Mouse Hematopoiesis

Sex dimorphism can be found in hematopoietic stem and progenitor cells, as well as in the development of hematopoietic lineages, resulting in sexual dimorphisms in normal hematopoiesis. Furthermore, males are at higher risk and have a worse prognosis for most hematologic malignancies than females. Under normal conditions, the number of circulating progenitor cells in women is lower than in men. Furthermore, female mice have a higher rate of HSC division than male mice. However, the molecular mechanism underlying normal hematopoiesis sex dimorphism is largely unknown. We compared male and female hematopoiesis in C57BL/6 mice in the steady state and discovered that male mice have more HSCs than female mice. Female LSK (Lineage-, Sca-1+, and cKit+) cells also had higher apoptosis and lower quiescence than male LSK cells. Additionally, male mice had higher percentage and number of mesenchymal stem cells (MSC) than female mice. We then conducted two sex/gender mismatch transplantation experiments. First, we injected male or female BM cells into male or female recipients, resulting in four transplantation groups (male to male, male to female, female to male, and female to female). We found that both male and female donor cells engraft better in male recipients than in female recipients, suggesting that male niche provides better hematopoietic engraftment. In the secondary transplantation setting, we mixed equal numbers of male (CD45.1) and female (CD45.2) BM cells and injected the cells into male and female CD45.1/CD45.2 chimeric recipient mice. Male donor cells engraft better than female donor cells in both male and female recipients. Consistently, male recipients have more support for HSC regeneration than female recipients. Altogether, these data suggest that male donor to male recipients produce the best engraftment outcome. This is consistent with the clinic bone marrow transplantation result. To better understand the underlying molecular mechanism, we performed single-cell RNA sequencing on LSK cells and bone marrow stromal cells from female and male mice (Lineage-, CD45-, Ter119-, and CD71-). Based on gene expression profiling at the single cell level, different populations of HSPCs and stromal cells were clustered. Mesenchymal stem cells (MSC) are the niche cellular component with the most significant sex difference, with male mice having more MSCs than female mice. The LSK cells were then co-cultured with male and female MSCs, and the cobblestone colony formation (CAFC) was assessed over time. The number of CAFC day 35 colonies was significantly higher in male MSC stromal than that in female MSCs. Therefore, male MSCs can thus better support for HSC function, which is consistent with our sex mismatch transplantation result. We further discovered that male MSC secrete more Cxcl12, which may underlie the increased hematopoietic regeneration. We are currently investigating the mechanism underlying Cxcl12 differential expression in the male/female niche by focusing on the Kdm5c gene. Kdm5c is a gene on the X chromosome that has escaped Xist inactivation. Female cells therefore express twice as much as male cells. It specifically demethylates H3K4me3/me2 and inhibits gene transcription. We hypothesize that a low level of Kdm5c in male MSCs leads to more methylation of H3K4me3, thus increasing Cxcl12 transcription. By using Chip-qPCR, we found that Kdm5c was highly enriched in the Cxcl12 promoter in female MSCs compared to male MSCs, leading to the increased level of demethylated H3K4me3, thus reducing expression of Cxcl12. We next knockdown Kdm5c in female MSCs and found that methylated H3K4me3 was increased in the Cxcl12 promotor, resulting in increased Cxcl12 expression. In conclusion, sex dimorphism affects normal hematopoietic in a cell intrinsic and extrinsic mechanism. Male MSCs provide a better support for hematopoietic engraftment and regeneration. Differential expression of Kdm5c, sex chromosome-specific gene, may be the underlying mechanism in which a lower level of KDM5C in male MSC leads to higher expression of Cxcl12, which promotes HSC engraftment and maintenance.

Discovering cell-active BCL6 inhibitors: effectively combining biochemical HTS with multiple biophysical techniques, X-ray crystallography and cell-based assays

By suppressing gene transcription through the recruitment of corepressor proteins, B-cell lymphoma 6 (BCL6) protein controls a transcriptional network required for the formation and maintenance of B-cell germinal centres. As BCL6 deregulation is implicated in the development of Diffuse Large B-Cell Lymphoma, we sought to discover novel small molecule inhibitors that disrupt the BCL6-corepressor protein–protein interaction (PPI). Here we report our hit finding and compound optimisation strategies, which provide insight into the multi-faceted orthogonal approaches that are needed to tackle this challenging PPI with small molecule inhibitors. Using a 1536-well plate fluorescence polarisation high throughput screen we identified multiple hit series, which were followed up by hit confirmation using a thermal shift assay, surface plasmon resonance and ligand-observed NMR. We determined X-ray structures of BCL6 bound to compounds from nine different series, enabling a structure-based drug design approach to improve their weak biochemical potency. We developed a time-resolved fluorescence energy transfer biochemical assay and a nano bioluminescence resonance energy transfer cellular assay to monitor cellular activity during compound optimisation. This workflow led to the discovery of novel inhibitors with respective biochemical and cellular potencies (IC50s) in the sub-micromolar and low micromolar range.

OR19-1 Ncor1 Regulates the Expression of Dhcr24, which Controls de novo Cholesterol Synthesis in the Adrenal Gland Inner Cortex

Abstract In many types of cells and tissues, the nuclear receptor corepressor 1 (Ncor1) is known as a main corepressor that interacts with thyroid hormone receptors (i.e., TRα, TRβ1, and TRβ2). In the absence of thyroid hormone (T3), NCOR1 is recruited by TRs to inhibit gene transcription. In the liver, the deletion of Ncor1 increases hepatic triglyceride levels. However, the detailed mechanism of how NCOR1 is involved in thyroid hormone signaling in different types of tissues is not fully understood. Our previous study showed that TRβ1 is expressed in the adrenal gland inner cortex and T3 treatment leads to the hypertrophy of this area. To determine the role of NCOR1 in the regulation of thyroid hormone signaling in adrenocortical cells, we generated the mice with a conditional deletion of Ncor1 in the adrenal cortex by crossing the Ncor1 floxed mice with the steroidogenic factor 1 (SF1) Cre mice. The RNA-seq data showed that only 76 genes were differentially expressed in adrenal glands due to the deletion of Ncor1 in adrenocortical cells. Gene ontology (GO) analysis revealed that 'cholesterol synthesis’ was the top GO term associated with these 76 genes. Interestingly, 24-dehydrocholesterol reductase (Dhcr24), the key enzyme for cholesterol synthesis, was upregulated in knockout mice adrenal glands in both sexes. This phenotype fits the results in our previous studies that (1) Dhcr24 is significantly increased under T3 treatment in wild-type mice adrenal glands and (2) the DHCR24-expressing zone in the inner cortex is the primary responsive adrenal cortical layer under T3 treatment which leads to increased lipid accumulation in this area. To further study how DHCR24 is involved in this T3-induced lipid accumulation in the adrenal gland inner cortex, we generated Dhcr24 conditional knockout mice. We found that the T3-induced lipid accumulation in the adrenal gland inner cortex was sharply decreased in the Dhcr24;Sf1-Cre mice. In summary, with the Ncor1 conditional knockout mice, we found that the regulation of the cholesterol synthesis through DHCR24 is the primary function of NCOR1 in the adrenal gland inner cortex; by using the Dhcr24 conditional knockout mice, we further confirmed that the T3-induced lipid accumulation in the adrenal inner cortex can be blocked by the deletion of DHCR24. This study used genetic mouse models to decipher the connection between nuclear receptor corepressor Ncor1, the cholesterol synthesis enzyme Dhcr24, and how they control de novo cholesterol synthesis in the adrenal gland inner cortex. Presentation: Monday, June 13, 2022 11:00 a.m. - 11:15 a.m.