Critical Transcriptional Regulator Research Articles

Discovery of a Potent, selective and orally bioavailable CDK9 degrader for targeting transcription regulation in Triple-Negative breast cancer

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous and invasive subtype of breast cancer with very limited effective modalities of treatment. Degrading the critical transcription regulator cyclin-dependent kinase 9 (CDK9) by proteolysis targeting chimeras (PROTACs) has shown promising potential for treating TNBC. However, to date, CDK9-targeting PROTACs for oral administration in treatment of cancers have not been reported. We herein present the design, synthesis, and extensive biological evaluation of a series of novel PROTACs as orally bioavailable, potent and selective degraders of CDK9 for targeting transcription regulation in triple-negative breast cancer. The developed compound 29 exhibited a desired potency (DC50 = 3.94 nM) with high efficacy (Dmax = 96 %) on CDK9 degradation, and effectively inhibited the proliferation of TNBC MDA-MB-231 cells. Mechanistic investigations revealed that compound 29 is a bona fide CDK9 degrader and can substantially downregulate the downstream targets c-Myc and MCL-1. Furthermore, compound 29 displayed favorable oral bioavailability in mice, and oral administration of degrader 29 significantly depleted CDK9 protein in TNBC tumor tissues and exhibited tumor growth inhibition in TNBC xenograft mice models. Collectively, our work established that degrader 29 is a highly potent and selective degraders of CDK9 with satisfactory oral bioavailability, which holds promising potential for the treatment of TNBC.

TetR-like regulator BP1026B_II1561 controls aromatic amino acid biosynthesis and intracellular pathogenesis in Burkholderia pseudomallei.

Burkholderia pseudomallei (Bp) causes the tropical disease melioidosis that afflicts an estimated 165,000 people each year. Bp is a facultative intracellular pathogen that transits through distinct intracellular stages including attachment to host cells, invasion through the endocytic pathway, escape from the endosome, replication in the cytoplasm, generation of protrusions towards neighboring cells, and host cell fusion allowing Bp infection to spread without exiting the intracellular environment. We have identified a TetR-like transcriptional regulator, BP1026B_II1561, that is up-regulated during the late stages of infection as Bp protrudes toward neighboring cells. We have characterized BP1026B_II1561 and determined that it has a role in pathogenesis. A deletional mutant of BP1026B_II1561 is attenuated in RAW264.7 macrophage and BALB/c mouse models of infection. Using RNA-seq, we found that BP1026B_II1561 controls secondary metabolite biosynthesis, fatty acid degradation, and propanoate metabolism. In addition, we identified that BP1026B_II1561 directly controls expression of an outer membrane porin and genes in the shikimate biosynthetic pathway using ChIP-seq. Transposon mutants of genes within the BP1026B_II1561 regulon show defects during intracellular replication in RAW264.7 cells confirming the role of this transcriptional regulator and the pathways it controls in pathogenesis. BP1026B_II1561 also up-regulates the majority of the enzymes in shikimate and tryptophan biosynthetic pathways, suggesting their importance for Bp physiology. To investigate this, we tested fluorinated analogs of anthranilate and tryptophan, intermediates and products of the shikimate and tryptophan biosynthetic pathways, respectively, and showed inhibition of Bp growth at nanomolar concentrations. The expression of these pathways by BP1026b_II1561 and during intracellular infection combined with the inhibition of Bp growth by fluorotryptophan/anthranilate highlights these pathways as potential targets for therapeutic intervention against melioidosis. In the present study, we have identified BP1026B_II1561 as a critical transcriptional regulator for Bp pathogenesis and partially characterized its role during host cell infection.

Hippo signaling pathway regulates Ebola virus transcription and egress

Filovirus-host interactions play important roles in all stages of the virus lifecycle. Here, we identify LATS1/2 kinases and YAP, key components of the Hippo pathway, as critical regulators of EBOV transcription and egress. Specifically, we find that when YAP is phosphorylated by LATS1/2, it localizes to the cytoplasm (Hippo “ON”) where it sequesters VP40 to prevent egress. In contrast, when the Hippo pathway is “OFF”, unphosphorylated YAP translocates to the nucleus where it transcriptionally activates host genes and promotes viral egress. Our data reveal that LATS2 indirectly modulates filoviral VP40-mediated egress through phosphorylation of AMOTp130, a positive regulator of viral egress, but more surprisingly that LATS1/2 kinases directly modulate EBOV transcription by phosphorylating VP30, an essential regulator of viral transcription. In sum, our findings highlight the potential to exploit the Hippo pathway/filovirus axis for the development of host-oriented countermeasures targeting EBOV and related filoviruses.

Human cytomegalovirus infection impairs neural differentiation via repressing sterol regulatory element binding protein 2-mediated cholesterol biosynthesis

Congenital human cytomegalovirus (HCMV) infection is a major cause of abnormalities and disorders in the central nervous system (CNS) and/or the peripheral nervous system (PNS). However, the complete pathogenesis of neural differentiation disorders caused by HCMV infection remains to be fully elucidated. Stem cells from human exfoliated deciduous teeth (SHEDs) are mesenchymal stem cells (MSCs) with a high proliferation and neurogenic differentiation capacity. Since SHEDs originate from the neural crest of the early embryonic ectoderm, SHEDs were hypothesized to serve as a promising cell line for investigating the pathogenesis of neural differentiation disorders in the PNS caused by congenital HCMV infection. In this work, SHEDs were demonstrated to be fully permissive to HCMV infection and the virus was able to complete its life cycle in SHEDs. Under neurogenic inductive conditions, HCMV infection of SHEDs caused an abnormal neural morphology. The expression of stem/neural cell markers was also disturbed by HCMV infection. The impairment of neural differentiation was mainly due to a reduction of intracellular cholesterol levels caused by HCMV infection. Sterol regulatory element binding protein-2 (SREBP2) is a critical transcription regulator that guides cholesterol synthesis. HCMV infection was shown to hinder the migration of SREBP2 into nucleus and resulted in perinuclear aggregations of SREBP2 during neural differentiation. Our findings provide new insights into the prevention and treatment of nervous system diseases caused by congenital HCMV infection.

Cadmium targeting transcription factor EB to inhibit autophagy-lysosome function contributes to acute kidney injury

IntroductionEnvironmental and occupational exposure to cadmium (Cd) has been shown to cause acute kidney injury (AKI). Previous studies have demonstrated that autophagy inhibition and lysosomal dysfunction are important mechanisms of Cd-induced AKI. ObjectivesTranscription factor EB (TFEB) is a critical transcription regulator that modulates autophagy-lysosome function, but its role in Cd-induced AKI is yet to be elucidated. Thus, in vivo and in vitro studies were conducted to clarify this issue. Methods and resultsData firstly showed that reduced TFEB expression and nuclear translocation were evident in Cd-induced AKI models, accompanied by autophagy-lysosome dysfunction. Pharmacological and genetic activation of TFEB improved Cd-induced AKI via alleviating autophagy inhibition and lysosomal dysfunction, whereas Tfeb knockdown further aggravated this phenomenon, suggesting the key role of TFEB in Cd-induced AKI by regulating autophagy. Mechanistically, Cd activated mechanistic target of rapamycin complex 1 (mTORC1) to enhance TFEB phosphorylation and thereby inhibiting TFEB nuclear translocation. Cd also activated chromosome region maintenance 1 (CRM1) to promote TFEB nuclear export. Meanwhile, Cd activated general control non-repressed protein 5 (GCN5) to enhance nuclear TFEB acetylation, resulting in the decreased TFEB transcriptional activity. Moreover, inhibition of CRM1 or GCN5 alleviated Cd-induced AKI by enhancing TFEB activity, respectively. ConclusionIn summary, these findings reveal that TFEB phosphorylation, nuclear export and acetylation independently suppress TFEB activity to cause Cd-induced AKI via regulating autophagy-lysosome function, suggesting that TFEB activation might be a promising treatment strategy for Cd-induced AKI.

MODL-14. TARGETING GL1/GL2-DNMT1-UHRF1 EPIGENETIC COMPLEX DISRUPTION VIA BET INHIBITION IMPAIRS SHH MB DEVELOPMENT

Abstract BACKGROUND Sonic Hedgehog Medulloblastoma (SHH MB) shares 30% among all molecular subtypes of medulloblastomas (MBs). Constitutive activation of SHH signaling in the cerebellum’s granular cell precursors (GCPs) is critical for this MB development. When SHH receptor PATCHED-1 (PTCH1) is mutated, stimulation of this receptor by SHH ligand binding leads to derepression of heterotrimeric GTP-binding protein–coupled receptor Smoothened (SMO) eventually altering the activity, location, and stability of the three downstream glioma associated oncogene (GLI) family members: GLI1, GLI2, and GLI3. GLI1 and GLI2 mostly function as transcriptional activators, while GLI3 mainly plays a repressor role. The bromodomain and extra-terminal domain (BET) family of proteins comprises four conserved members (Brd2, Brd3, Brd4, and Brdt), which are critical regulators of GLI1 and GLI2-mediated transcription. METHODS In the present study, we pharmacologically inhibited BETs with a previously described BET inhibitor, BMS986158, and a novel BET inhibitor, UM002, discovered by our lab. We treated human SHH MB cells (ONS76 cells) in vitro and ex vivo in mouse SHH MB cells (from Ptch1 conditional knockout mice) with minimum effective concentrations of these inhibitors. We screened for GLI1/GLI2-DNMT1-UHRF1 epigenetic complex components expressions by immunoblotting and assessed the effectiveness of inhibiting their interactions by performing PLA assays. RESULTS In this study, we show that pharmacological inhibition of BETs leads to attenuated GLI1 and GLI2 levels. This GLI1 and GLI2 inhibition eventually disrupts the previously reported GL1/GL2, DNMT1-UHRF1 epigenetic complex, which is crucial for SHH MB development. A previously described BET inhibitor, BMS986158, and a novel BET inhibitor, UM002, synthesized and characterized by our lab can inhibit BETs. This could be a potential therapeutic approach targeting GLI-associated epigenetic complexes to treat SHH medulloblastoma.

Abstract 104: Endothelial Restoration Of CAD GWAS Gene PLPP3 By Nanomedicine Suppresses Mechano-sensitive YAP/TAZ Activity And Reduces Atherosclerosis In Vivo

Genome-wide association studies (GWAS) have suggested new molecular mechanisms driving vascular complications such as coronary artery disease (CAD). Nevertheless, a major challenge to developing new therapeutic approaches is to spatiotemporally manipulate these GWAS-identified genes in specific vascular tissues of interest in vivo . Atherosclerosis largely occurs in arterial sites where local disturbed flow (DF) activates vascular endothelial cells. The Yorkie homologues YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) have merged as critical transcriptional regulators responding to biomechanical stimuli. The molecular mechanisms by which DF activates while unidirectional flow (UF) inactivates YAP/TAZ remain incompletely understood. Our recent studies demonstrated that DF and genetic predisposition at the CAD locus 1p32.2 cooperatively suppress endothelial phospholipid phosphatase 3 (PLPP3) transcription by inactivating a mechano-sensitive endothelial enhancer; reduced PLPP3 and consequent activation of lipid lysophosphatidic acid (LPA)-receptor signaling, drive endothelial dysfunction. Here we devised a nanomedicine strategy employing VCAM-1-targeting liposome to preferentially deliver chemically-modified PLPP3 mRNA to inflamed endothelial cells in vivo , effectively suppressing endothelial YAP/TAZ activity and reducing DF-induced atherosclerosis in mice. We discovered a molecular paradigm by which PLPP3 inactivates YAP/TAZ by reducing LPA-induced myosin II and ROCK1. These results highlight a new mechanistic link between GWAS and YAP/TAZ mechano-regulation and moreover, establish the proof of concept of vascular wall-based therapies employing targeted mRNA-based nanomedicine.

Chromatin profiling reveals TFAP4 as a critical transcriptional regulator of bovine satellite cell differentiation

BackgroundSatellite cells are myogenic precursor cells in adult skeletal muscle and play a crucial role in skeletal muscle regeneration, maintenance, and growth. Like embryonic myoblasts, satellite cells have the ability to proliferate, differentiate, and fuse to form multinucleated myofibers. In this study, we aimed to identify additional transcription factors that control gene expression during bovine satellite cell proliferation and differentiation.ResultsUsing chromatin immunoprecipitation followed by sequencing, we identified 56,973 and 54,470 genomic regions marked with both the histone modifications H3K4me1 and H3K27ac, which were considered active enhancers, and 50,956 and 59,174 genomic regions marked with H3K27me3, which were considered repressed enhancers, in proliferating and differentiating bovine satellite cells, respectively. In addition, we identified 1,216 and 1,171 super-enhancers in proliferating and differentiating bovine satellite cells, respectively. Analyzing these enhancers showed that in proliferating bovine satellite cells, active enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation whereas repressed enhancers were associated with genes essential for myoblast differentiation, and that in differentiating satellite cells, active enhancers were associated with genes essential for myoblast differentiation or muscle contraction whereas repressed enhancers were associated with genes stimulating cell proliferation or inhibiting myoblast differentiation. Active enhancers in proliferating bovine satellite cells were enriched with binding sites for many transcription factors such as MYF5 and the AP-1 family transcription factors; active enhancers in differentiating bovine satellite cells were enriched with binding sites for many transcription factors such as MYOG and TFAP4; and repressed enhancers in both proliferating and differentiating bovine satellite cells were enriched with binding sites for NF-kB, ZEB-1, and several other transcription factors. The role of TFAP4 in satellite cell or myoblast differentiation was previously unknown, and through gene knockdown and overexpression, we experimentally validated a critical role for TFAP4 in the differentiation and fusion of bovine satellite cells into myofibers.ConclusionsSatellite cell proliferation and differentiation are controlled by many transcription factors such as AP-1, TFAP4, NF-kB, and ZEB-1 whose roles in these processes were previously unknown in addition to those transcription factors such as MYF5 and MYOG whose roles in these processes are widely known.

Mechanotransduction-induced interplay between phospholamban and yes-activated protein induces smooth muscle cell hypertrophy

The gastrointestinal system is a hollow organ affected by fibrostenotic diseases that cause volumetric compromise of the lumen via smooth muscle hypertrophy and fibrosis. Many of the driving mechanisms remain unclear. Yes-associated protein-1 (YAP) is a critical mechanosensory transcriptional regulator that mediates cell hypertrophy in response to elevated extracellular rigidity. In the type 2 inflammatory disorder, eosinophilic esophagitis (EoE), phospholamban (PLN) can induce smooth muscle cell hypertrophy. We used EoE as a disease model for understanding a mechanistic pathway in which PLN and YAP interact in response to rigid extracellular substrate to induce smooth muscle cell hypertrophy. PLN induced YAP nuclear sequestration in a feed forward loop that caused increased cell size in response to rigid substrate. This mechanism of rigidity sensing may have previously unappreciated clinical implications for PLN expressing hollow systems such as the esophagus and heart.

Abstract 11679: The Crucial Role of Estrogen-Related Receptor in Human Cardiomyocyte Maturation

During the developmental transition from fetal to adult, the heart undergoes dramatic mitochondrial and sarcomeric maturation. This process is partially reversed during development of heart failure. We have identified the nuclear receptors, estrogen-related receptor (ERR) α and γ, as critical transcriptional regulators of energy metabolic and sarcomeric genes during postnatal maturation of mouse heart. Here, we aimed to define the transcriptional mechanisms involved in ERR-mediated maturation in human cardiomyocytes. Genomic interrogation with published datasets revealed that approximately 50% of super-enhancer regions (SEs) overlapped with ERRγ peaks in human-induced pluripotent stem cell-derived cardiac myocytes. Intersection of the ERRγ cistrome with a published H3K27ac Hi-ChIP dataset from human hearts identified over 3,000 ERR-containing enhancer-promoter loops, which are associated with genes related to cardiac contractile function. These results highlighted the essential role of ERR in cardiomyocyte enhancer function. Among the SEs defined by mediator complex deposition, we found that both ERRα and γ occupied the enhancer region flanking the MYH6/MYH7 cluster, a well-characterized locus for fetal to adult developmental switching. Targeting ERR recruitment on the enhancer using catalytically-dead Cas9-KRAB or proteolysis targeting chimeric (PROTAC) strategies resulted in the reversal of MYH6/7 switching. RNA-sequencing analysis following PROTAC treatment revealed that deactivation of ERRα resulted in downregulation of a wide array of genes encoding cardiac structural proteins, particularly adult-type ventricular sarcomeric proteins. Additional cistrome analyses demonstrated that PROTAC treatment resulted in decreased ERRα peak signals on cardiac structural genes, including enhancer regions directing MYH6/7 transcription consistent with the observed gene expression changes. In summary, ERR signaling is essential for the transcriptional program of structural maturation in human cardiomyocytes. This process likely involves establishing proper cardiac chromatin architecture. These results support a novel therapeutic strategy of utilizing ERR activators to reverse fetal shifts in failing hearts.

Bioinformatics analysis of competing endogenous RNA network in decidual natural killer cell from unexplained recurrent spontaneous abortion.

Decidual natural killer (dNK) cell plays a pivotal role in maintaining pregnancy, especially in the first trimester. Noncoding-RNAs (ncRNAs) are critical regulators of transcription and protein expression. Dysregulation of ncRNAs may be involved in the pathogenesis of unexplained recurrent spontaneous abortion (URSA). However, the role of competing endogenous RNA (ceRNA) based on mRNA-miRNA-lncRNA network in regulating the incidence and progression of URSA remains elusive. The aim of the study is to identify the regulatory network of mRNA-miRNA-LncRNA ceRNA based on bioinformatics analysis in dNK from patients with URSA. Eligible studies were retrieved from PubMed, Embase, and the Gene Expression Omnibus (GEO) databases to identify differentially expressed genes (DEGs), miRNAs and LncRNAs in dNK cells of patients with URSA. Protein-protein interaction (PPI) network was constructed by STRING database and Cytoscape software. Potential regulatory miRNAs and lncRNAs of mRNAs were predicted by miRTarBase and RNA22 and subject to bioinformatics analysis. A total of 634 DEGs were screened, including 290 upregulated and 344 downregulated DEGs. Among 207 differentially expressed lncRNAs, 110 lncRNAs were upregulated and 97 were downregulated. According to node degree, 30 hub genes were identified for subsequent research. After drawing the Venn diagram and matching to Cytoscape, an mRNA-miRNA-lncRNA network linked to the pathogenesis of URSA in dNK cells was constructed. A novel regulatory network of mRNA-miRNA-lncRNA ceRNA is established in dNK cells from patients with URSA. All RNAs might be used as the biomarkers of the pathogenesis of URSA.

Role of Non-Coding RNAs in TGF-β Signalling in Glioma.

Brain tumours and Gliomas, in particular, are among the primary causes of cancer mortality worldwide. Glioma diagnosis and therapy have not significantly improved despite decades of efforts. Autocrine TGF-β signalling promotes glioma proliferation, invasion, epithelial-to-mesenchymal transition (EMT), and drug resistance. Non-coding RNAs such as miRNA, lncRNA, and circRNAs have emerged as critical transcriptional and post-transcriptional regulators of TGF-β pathway components in glioma. Here, we summarize the complex regulatory network among regulatory ncRNAs and TGF-β pathway during Glioma pathogenesis and discuss their role as potential therapeutic targets for Gliomas.

TCF4 Mutations Disrupt Synaptic Function Through Dysregulation of RIMBP2 in Patient-Derived Cortical Neurons

BackgroundGenetic variation in the TCF4 (transcription factor 4) gene is associated with risk for a variety of developmental and psychiatric conditions, which includes a syndromic form of autism spectrum disorder called Pitt-Hopkins syndrome (PTHS). TCF4 encodes an activity-dependent transcription factor that is highly expressed during cortical development and in animal models has been shown to regulate various aspects of neuronal development and function. However, our understanding of how disease-causing mutations in TCF4 confer pathophysiology in a human context is lacking. MethodsTo model PTHS, we differentiated human cortical neurons from human induced pluripotent stem cells that were derived from patients with PTHS and neurotypical individuals. To identify pathophysiology and disease mechanisms, we assayed cortical neurons with whole-cell electrophysiology, Ca2+ imaging, multielectrode arrays, immunocytochemistry, and RNA sequencing. ResultsCortical neurons derived from patients with TCF4 mutations showed deficits in spontaneous synaptic transmission, network excitability, and homeostatic plasticity. Transcriptomic analysis indicated that these phenotypes resulted in part from altered expression of genes involved in presynaptic neurotransmission and identified the presynaptic binding protein RIMBP2 as the most differentially expressed gene in PTHS neurons. Remarkably, TCF4-dependent deficits in spontaneous synaptic transmission and network excitability were rescued by increasing RIMBP2 expression in presynaptic neurons. ConclusionsTaken together, these results identify TCF4 as a critical transcriptional regulator of human synaptic development and plasticity and specifically identifies dysregulation of presynaptic function as an early pathophysiology in PTHS.

MetaTiME integrates single-cell gene expression to characterize the meta-components of the tumor immune microenvironment

Recent advances in single-cell RNA sequencing have shown heterogeneous cell types and gene expression states in the non-cancerous cells in tumors. The integration of multiple scRNA-seq datasets across tumors can indicate common cell types and states in the tumor microenvironment (TME). We develop a data driven framework, MetaTiME, to overcome the limitations in resolution and consistency that result from manual labelling using known gene markers. Using millions of TME single cells, MetaTiME learns meta-components that encode independent components of gene expression observed across cancer types. The meta-components are biologically interpretable as cell types, cell states, and signaling activities. By projecting onto the MetaTiME space, we provide a tool to annotate cell states and signature continuums for TME scRNA-seq data. Leveraging epigenetics data, MetaTiME reveals critical transcriptional regulators for the cell states. Overall, MetaTiME learns data-driven meta-components that depict cellular states and gene regulators for tumor immunity and cancer immunotherapy.

Abstract LB134: Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth

Abstract Background: In-frame chimeric proteins that are encoded by ESR1 exons 1 to 6 (ESR1-e6) fused to C-terminal gene partners can induce hormone-independent growth and endocrine therapy (ET) resistance of estrogen receptor-α (ERα) positive breast cancer by driving a unique transcriptional signature (PMID: 34711608). However, the mechanism how active ESR1 fusions reprogram transcription is not understood. Moreover, the lack of targeted treatment due to the loss of ligand binding domain (LBD) in ESR1 gene fusions necessitates the need to develop new therapies targeting active ESR1 fusions that promote tumorigenesis. Methods: An estrogen response element (ERE) DNA pull down assay was performed to identify coactivators recruited by active ESR1 fusions. An alamar blue assay was utilized to assess cell growth after treatment with pharmacological inhibitors to the coactivators. Single cells isolated from ERα+ patient-derived xenografts (PDX) were used to form organoids (PDxOs). A CellTiter-Glo 3D assay was conducted to measure the growth of PDxOs after treatment with coactivator inhibitors. Results: Using ERE DNA pull down assays, we identified bromodomain-containing proteins (BRDs) and steroid receptor coactivators (SRCs/p160 family members) as key regulators recruited by active ESR1 fusions from nuclear extracts of breast cancer cells. A pan-BET family inhibitor (BETi), birabresib, and pan-SRC inhibitors (SRCi), SI-2 and SI-12, inhibited the growth of ERα positive breast cancer cells and PDxOs expressing an active ESR1 fusion or LBD point mutation. Importantly, the combination of birabresib and a CDK4/6 kinase inhibitor (palbociclib) conferred the largest growth reduction of PDxOs expressing an ESR1-YAP1 fusion. RNA sequencing showed BETi and SRCi led to downregulation of ERα target gene expression and cell cycle pathways, with upregulation of apoptosis pathways. Here, we propose a model whereby BRD and SRC protein families are major regulators of active ESR1 fusion-driven gene transcription and growth and positioned the potential therapeutic significance of BETi and SRCi to be tailored to treat active ESR1-translocated breast tumors. Conclusions: Active ESR1 fusions cannot be treated with standard-of-care ET due to the lack of the LBD of ERα. Therefore, it is critical to investigate how active, in-frame ESR1 fusion proteins induce gene transcription and to develop molecular mechanism-based targeted therapies. Here, we revealed two groups of coactivators (BRDs and SRCs) as critical transcriptional regulators of active ESR1 fusion-driven tumor biology. Our study demonstrated the efficacy of a BETi (birabresib) and SRCi (SI-2/SI-12) in inhibiting the viability of active ESR1 fusion/LBD mutation expressing breast cancer cells and 3D grown PDxOs. Importantly, birabresib combined with a standard-of-care CDK4/6 inhibitor conferred the best PDxO growth reduction. Citation Format: Xuxu Gou, Craig T. Vollert, Yue Chen, Diana Fandino, Matthew V. Holt, Beom-Jun Kim, Qin Feng, Jin Wang, David M. Lonard, Bert W. O'Malley, Matthew J. Ellis, Charles E. Foulds. Targeting coactivators to inhibit ESR1 fusion-driven breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB134.

METTL5 stabilizes c-Myc by facilitating USP5 translation to reprogram glucose metabolism and promote hepatocellular carcinoma progression.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers in the world, with a high likelihood of metastasis and a dismal prognosis. The reprogramming of glucose metabolism is critical in the development of HCC. The Warburg effect has recently been confirmed to occur in a variety of cancers, including HCC. However, little is known about the molecular biological mechanisms underlying the Warburg effect in HCC cells. In this study, we sought to better understand how methyltransferase 5, N6-adenosine (METTL5) controls the development of HCC and the Warburg effect. In the current study, quantitative real-time polymerase chain reaction and Western blotting were used to detect the expression of METTL5 in HCC tissues and cell lines. Several different cell models and animal models were established to determine the role of METTL5 in glucose metabolism reprogramming and the underlying molecular mechanism of HCC. Glutathione-S-transferase pulldown, coimmunoprecipitation, RNA sequencing, non-targeted metabolomics, polysome profiling, and luciferase reporter assays were performed to investigate the molecular mechanisms of METTL5 in HCC cells. We discovered that METTL5 drove glucose metabolic reprogramming to promote the proliferation and metastasis of HCC. Mechanistically, upregulation of METTL5 promoted c-Myc stability and thus activated its downstream glycolytic genes lactate dehydrogenase A (LDHA), enolase 1 (ENO1), triosephosphate isomerase 1 (TPI1), solute carrier family 2 member 1 (SLC2A1), and pyruvate kinase M2 (PKM2). The c-Box and ubiquitin binding domain (UBA) regions of ubiquitin specific peptidase 5 (USP5) binded to c-Myc protein and inhibited K48-linked polyubiquitination of c-Myc. Further study revealed that METTL5 controled the USP5 translation process, which in turn regulated the ubiquitination of c-Myc. Furthermore, we identified cAMP responsive element binding protein 1 (CREB1)/P300 as a critical transcriptional regulator of METTL5 that promoted the transcription of METTL5 in HCC. In patient-derived tumor xenograft (PDX) models, adenovirus-mediated knockout of METTL5 had a good antitumor effect and prolonged the survival of PDX-bearing mice. These findings point to a novel mechanism by which CREB1/P300-METTL5-USP5-c-Myc controls abnormal glucose metabolism and promotes tumor growth, suggesting that METTL5 is a potential therapeutic target and prognostic biomarker for HCC.

TREM-1 governs NLRP3 inflammasome activation of macrophages by firing up glycolysis in acute lung injury.

The triggering receptor expressed on myeloid cells-1 (TREM-1) is a pro-inflammatory immune receptor potentiating acute lung injury (ALI). However, the mechanism of TREM-1-triggered inflammation response remains poorly understood. Here, we showed that TREM-1 blocking attenuated NOD-, LRR- and pyrin domain-containing 3 (NLRP3) inflammasome activation and glycolysis in LPS-induced ALI mice. Then, we observed that TREM-1 activation enhanced glucose consumption, induced glycolysis, and inhibited oxidative phosphorylation in macrophages. Specifically, inhibition of glycolysis with 2-deoxyglucose diminished NLRP3 inflammasome activation of macrophages triggered by TREM-1. Hypoxia-inducible factor-1α (HIF-1α) is a critical transcriptional regulator of glycolysis. We further found that TREM-1 activation facilitated HIF-1α accumulation and translocation to the nucleus via the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. Inhibiting mTOR or HIF-1α also suppressed TREM-1-induced metabolic reprogramming and NLRP3/caspase-1 activation. Overall, the mTOR/HIF-1α/glycolysis pathway is a novel mechanism underlying TREM-1-governed NLRP3 inflammasome activation. Therapeutic targeting of the mTOR/HIF-1α/glycolysis pathway in TREM-1-activated macrophages could be beneficial for treating or preventing inflammatory diseases, such as ALI.

PRDM10 directs FLCN expression in a novel disorder overlapping with Birt-Hogg-Dubé syndrome and familial lipomatosis.

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder characterized by fibrofolliculomas, pulmonary cysts, pneumothoraces and renal cell carcinomas. Here, we reveal a novel hereditary disorder in a family with skin and mucosal lesions, extensive lipomatosis and renal cell carcinomas. The proband was initially diagnosed with BHD based on the presence of fibrofolliculomas, but no pathogenic germline variant was detected in FLCN, the gene associated with BHD. By whole exome sequencing we identified a heterozygous missense variant (p.(Cys677Tyr)) in a zinc-finger encoding domain of the PRDM10 gene which co-segregated with the phenotype in the family. We show that PRDM10Cys677Tyr loses affinity for a regulatory binding motif in the FLCN promoter, abrogating cellular FLCN mRNA and protein levels. Overexpressing inducible PRDM10Cys677Tyr in renal epithelial cells altered the transcription of multiple genes, showing overlap but also differences with the effects of knocking out FLCN. We propose that PRDM10 controls an extensive gene program and acts as a critical regulator of FLCN gene transcription in human cells. The germline variant PRDM10Cys677Tyr curtails cellular folliculin expression and underlies a distinguishable syndrome characterized by extensive lipomatosis, fibrofolliculomas and renal cell carcinomas.

STEM-18. EPIGENETIC AND MOLECULAR COORDINATION BETWEEN HDAC2 AND SMAD3-SKI IS REQUIRED FOR GROWTH AND STEM CELL CHARACTERISTICS OF BRAIN TUMOUR STEM CELLS

Abstract Brain tumour stem cell population in glioblastoma (GBM) display key cancer stem cell characteristics of high self-renewal and drug resistance that are maintained by the coordinated functions of epigenetic and molecular regulators. Yet, specific epigenetic mechanisms that, in collaboration with relevant molecular pathways, help maintain a stem-like state in BTSCs remain poorly understood. Here, we identify HDAC2 as a foremost epigenetic regulator in BTSCs that specifically utilizes the transforming growth factor-β (TGF-β) pathway related proteins, SMAD3-SKI, for remodelling BTSC chromatin accessibility and transcriptional programs to facilitate their stemness and tumorigenic potentials. Our initial drug screening revealed that selective inhibition of HDAC1 and 2 with romidepsin was effective in targeting BTSC viability, cell proliferation and self-renewal in vitro. Using CRISPR-cas9 knockout and shRNA knockdown strategies, we further demonstrated that loss of HDAC2 disrupts an epigenetic and molecular coordination between HDAC2 and SMAD-SKI proteins, which negatively impacts BTSC survival, cell proliferation and self-renewal in vitro and improves median survival in orthotopic xenograft mouse models. Loss of HDAC2 showed reduction in the protein abundance of transcriptional regulator, SMAD3 and negative regulator protein, SKI. However, overexpression of SMAD3 in HDAC2 deficient BTSCs could partially rescues their cell functional deficits. These findings suggest that context-specific epigenetic regulations by HDAC2 and its interaction with the critical transcriptional regulators, SMAD3-SKI, maintains the stemness and growth characteristics of BTSCs. Further HDAC2 overexpression increases cell proliferation and self-renewal abilities in normal neural stem cells (NSCs). These findings thus support the role of HDAC2 as a key epigenetic determinant of stemness in normal NSCs and of cancer stem cell characteristics and tumorigenic potential in BTSCs. Collectively, our data raises the potential that disruption of the coordinated mechanisms regulated by HDAC2-SMAD3-SKI axis may be an effective therapeutic approach for targeting GBM BTSCs.

PRMT3 drives glioblastoma progression by enhancing HIF1A and glycolytic metabolism

Glioblastoma (GBM) is the most common and aggressive primary brain tumor, but the mechanisms underlying tumor growth and progression remain unclear. The protein arginine methyltransferases (PRMTs) regulate a variety of biological processes, however, their roles in GBM growth and progression are not fully understood. In this study, our functional analysis of gene expression networks revealed that among the PRMT family expression of PRMT3 was most significantly enriched in both GBM and low-grade gliomas. Higher PRMT3 expression predicted poorer overall survival rate in patients with gliomas. Knockdown of PRMT3 markedly reduced the proliferation and migration of GBM cell lines and patient-derived glioblastoma stem cells (GSC) in cell culture, while its over-expression increased the proliferative capacity of GSC cells by promoting cell cycle progression. Consistently, stable PRMT3 knockdown strongly inhibited tumor growth in xenograft mouse models, along with a significant decrease in cell proliferation as well as an increase in apoptosis. We further found that PRMT3 reprogrammed metabolic pathways to promote GSC growth via increasing glycolysis and its critical transcriptional regulator HIF1α. In addition, pharmacological inhibition of PRMT3 with a PRMT3-specific inhibitor SGC707 impaired the growth of GBM cells. Thus, our study demonstrates that PRMT3 promotes GBM progression by enhancing HIF1A-mediated glycolysis and metabolic rewiring, presenting a point of metabolic vulnerability for therapeutic targeting in malignant gliomas.