Inducible Transcription Factor Research Articles

Inhibition of Survival Mechanisms and Cell Death Induction in Melanoma Following Photodynamic Therapy Mediated by Meso-5,10,15,20-tetrakis-(4-hydroxyphenyl)-porphyrin

(1) Background: Photodynamic therapy (PDT) involves the selective killing of tumor cells by the generation of reactive oxygen species using a photosensitizer (PS) activated by irradiation. In melanoma, PDT efficiency is altered by several mechanisms, such as the presence of melanin and melanosomes and pro-survival pathways mediated by transcription factors such as: AP-1 (activator protein), MITF (microphthalmia inducible transcription factor), HIF1α (hypoxia inducible factor), and NF-kB (nuclear factor kappa B). The study aimed to investigate the anti-melanoma effects of PDT mediated by meso-5,10,15,20-tetrakis-(4-hydroxyphenyl)-porphyrin (THPP) as a photosensitizer. (2) Methods: Cocultures of melanoma, two human, WM35 and M1–15, and murine B16-F10, with endothelial cells, were used. Cytotoxicity, oxidative damage, angiogenesis markers, and melanogenesis were assessed using colorimetry, flowcytometry, confocal microscopy, spectrophotometry, ELISA, and Western blotting. (3) Results: The maximal killing efficiency of PDT was reached in WM35, followed by M1–15, and then B16-F10, and it occurred through both apoptosis and necrosis. Although constitutive pigmentation diminished the PDT efficiency, de novo melanogenesis exhibited no protection. PDT increased TNFα, and inhibited NFkB, MITF, HIF1α, and AP1, leading to inflammation and angiogenesis markers’ inhibition. (4) Conclusions: THPP-mediated PDT efficiently induced cell death through apoptosis, necrosis, and the inhibition of pro-survival pathways mediated by NFkB, AP1, HIF1α, and MITF in the melanoma coculture models.

Chronic Neuronal Inactivity Utilizes the mTOR-TFEB Pathway to Drive Transcription-Dependent Autophagy for Homeostatic Up-Scaling.

Activity-dependent changes in protein expression are critical for neuronal plasticity, a fundamental process for the processing and storage of information in the brain. Among the various forms of plasticity, homeostatic synaptic up-scaling is unique in that it is induced primarily by neuronal inactivity. However, precisely how the turnover of synaptic proteins occurs in this homeostatic process remains unclear. Here, we report that chronically inhibiting neuronal activity in primary cortical neurons prepared from embryonic day (E)18 Sprague Dawley rats (both sexes) induces autophagy, thereby regulating key synaptic proteins for up-scaling. Mechanistically, chronic neuronal inactivity causes dephosphorylation of ERK and mTOR, which induces transcription factor EB (TFEB)-mediated cytonuclear signaling and drives transcription-dependent autophagy to regulate αCaMKII and PSD95 during synaptic up-scaling. Together, these findings suggest that mTOR-dependent autophagy, which is often triggered by metabolic stressors such as starvation, is recruited and sustained during neuronal inactivity to maintain synaptic homeostasis, a process that ensures proper brain function and if impaired can cause neuropsychiatric disorders such as autism.SIGNIFICANCE STATEMENT In the mammalian brain, protein turnover is tightly controlled by neuronal activation to ensure key neuronal functions during long-lasting synaptic plasticity. However, a long-standing question is how this process occurs during synaptic up-scaling, a process that requires protein turnover but is induced by neuronal inactivation. Here, we report that mTOR-dependent signaling, which is often triggered by metabolic stressors such as starvation, is "hijacked" by chronic neuronal inactivation, which then serves as a nucleation point for transcription factor EB (TFEB) cytonuclear signaling that drives transcription-dependent autophagy for up-scaling. These results provide the first evidence of a physiological role of mTOR-dependent autophagy in enduing neuronal plasticity, thereby connecting major themes in cell biology and neuroscience via a servo loop that mediates autoregulation in the brain.

Up-regulated Circular RNAs in Colorectal Cancer: New Entities for Therapy and Tools for Identification of Therapeutic Targets.

Patients with disseminated colorectal cancer have a dismal prognosis with a 5-year survival rate of only 13%. In order to identify new treatment modalities and new targets, we searched the literature for up-regulated circular RNAs in colorectal cancer which induce tumor growth in corresponding preclinical in vivo models. We identified nine circular RNAs that mediate resistance against chemotherapeutic agents, seven that up-regulate transmembrane receptors, five that induce secreted factors, nine that activate signaling components, five which up-regulate enzymes, six which activate actin-related proteins, six which induce transcription factors and two which up-regulate the MUSASHI family of RNA binding proteins. All of the circular RNAs discussed in this paper induce the corresponding targets by sponging microRNAs (miRs) and can be inhibited by RNAi or shRNA in vitro and in xenograft models. We have focused on circular RNAs with demonstrated activity in preclinical in vivo models because the latter is an important milestone in drug development. All circular RNAs with in vitro activity only data are not referenced in this review. The translational impact of inhibition of these circular RNAs and of the identified targets for treatment of colorectal cancer (CRC) are discussed.

Abstract P2-16-03: The Speckle-type POZ protein (SPOP) inhibits breast cancer malignancy by destabilizing oncogene TWIST1

Abstract Epithelial-mesenchymal transition (EMT) inducing transcription factor TWIST1 plays a vital role in cancer metastasis. How the tumor-suppressive E3 ligase, Speckle-type POZ protein (SPOP), regulates TWIST1 in breast cancer remains unknown. In this study, we report that SPOP physically interacts with, ubiquitinates, and destabilizes TWIST1. SPOP promotes K63-and K48-linked ubiquitination of TWIST1, predominantly at K73, thereby suppressing cancer cell migration and invasion. Silencing SPOP significantly enhances EMT, which accelerates breast cancer cell migration and invasiveness in vitro and lung metastasis in vivo. Clinically, SPOP is negatively correlated with the levels of TWIST1 in highly invasive breast carcinomas. Reduced SPOP expression, along with elevated TWIST1 levels, is associated with poor prognosis in advanced breast cancer patients, particularly those with metastatic triple-negative breast cancer (TNBC). Taken together, we have disclosed a new mechanism linking SPOP to TWIST1 degradation. Thus SPOP may serve as a prognostic marker and a potential therapeutic target for advanced TNBC patients. Citation Format: Chunli Wei, Yun Liu, Xiaoyan Liu, Jingliang Cheng, Jiewen Fu, Xiuli Xiao, Robb E Moses, Xiaotao Li, Junjiang Fu. The Speckle-type POZ protein (SPOP) inhibits breast cancer malignancy by destabilizing oncogene TWIST1 [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-03.

Inducible CRISPR Epigenome Systems Mimic Cocaine Induced Bidirectional Regulation of Nab2 and Egr3.

Substance use disorder is a chronic disease and a leading cause of disability around the world. The NAc is a major brain hub mediating reward behavior. Studies demonstrate exposure to cocaine is associated with molecular and functional imbalance in NAc medium spiny neuron subtypes (MSNs), dopamine receptor 1 and 2 enriched D1-MSNs and D2-MSNs. We previously reported repeated cocaine exposure induced transcription factor early growth response 3 (Egr3) mRNA in NAc D1-MSNs, and reduced it in D2-MSNs. Here, we report our findings of repeated cocaine exposure in male mice inducing MSN subtype-specific bidirectional expression of the Egr3 corepressor NGFI-A-binding protein 2 (Nab2). Using CRISPR activation and interference (CRISPRa and CRISPRi) tools combined with Nab2 or Egr3-targeted sgRNAs, we mimicked these bidirectional changes in Neuro2a cells. Furthermore, we investigated D1-MSN- and D2-MSN-specific expressional changes of histone lysine demethylases Kdm1a, Kdm6a, and Kdm5c in NAc after repeated cocaine exposure in male mice. Since Kdm1a showed bidirectional expression patterns in D1-MSNs and D2-MSNs, like Egr3, we developed a light-inducible Opto-CRISPR-KDM1a system. We were able to downregulate Egr3 and Nab2 transcripts in Neuro2A cells and cause similar bidirectional expression changes we observed in D1-MSNs and D2-MSNs of mouse repeated cocaine exposure model. Contrastingly, our Opto-CRISPR-p300 activation system induced the Egr3 and Nab2 transcripts and caused opposite bidirectional transcription regulations. Our study sheds light on the expression patterns of Nab2 and Egr3 in specific NAc MSNs in cocaine action and uses CRISPR tools to further mimic these expression patterns.SIGNIFICANCE STATEMENT Substance use disorder is a major societal issue. The lack of medication to treat cocaine addiction desperately calls for a treatment development based on precise understanding of molecular mechanisms underlying cocaine addiction. In this study, we show that Egr3 and Nab2 are bidirectionally regulated in mouse NAc D1-MSNs and D2-MSNs after repeated exposure to cocaine. Furthermore, histone lysine demethylations enzymes with putative EGR3 binding sites showed bidirectional regulation in D1- and D2-MSNs after repeated exposure to cocaine. Using Cre- and light-inducible CRISPR tools, we show that we can mimic this bidirectional regulation of Egr3 and Nab2 in Neuro2a cells.

The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition.

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor β (TGFβ) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional(3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFβ, including TGFβ auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFβ signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFβ type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFβ family members.

Integrative analyses of RNA-seq and ChIP-seq Reveal MITF as a Target Gene of TFPI-2 in MDA231 Cells.

Breast cancer is the most prevalent cancer in female patients worldwide. Tissue factor pathway inhibitor 2 (TFPI-2) is identified as an important tumor suppressor in various cancers. Recent studies have shown that TFPI-2 translocates into the nucleus, where it modulates the transcription of the matrix metalloproteinase-2 (MMP-2) gene. However, its biological role and molecular mechanisms in the progression of breast cancer remain unclear. In this study, we identified 5125 differentially expressed genes (DEGs) from RNA sequencing (RNA-seq) in TFPI-2-overexpressing MDA231 cells compared with control cells. Gene ontology and Kyoto encyclopedia of genes and genomes (KEGG) analysis shown that cell cycle, cell differentiation, proteoglycans in cancer, and pathways associated with cancer were highly enriched in downregulated DEGs. Integration of the RNA-seq and ChIP-sequencing (ChIP-seq) data identified 73 genes directly controlled by TFPI-2 in MDA231 cells. Among them, melanocyte inducing transcription factor (MITF) gene expression was repressed by TFPI-2, which was further verified by a luciferase reporter assay and ChIP-quantitative PCR. Our study provides evidence of a novel role of TFPI-2 in human breast cancer involving targeting of the MITF.

Lipids as Targets for Renal Cell Carcinoma Therapy

Kidney cancer is among the top ten most common cancers to date. Within the kidney, renal cell carcinoma (RCC) is the most common solid lesion occurring. While various risk factors are suspected, including unhealthy lifestyle, age, and ethnicity, genetic mutations seem to be a key risk factor. In particular, mutations in the von Hippel-Lindau gene (Vhl) have attracted a lot of interest since this gene regulates the hypoxia inducible transcription factors HIF-1α and HIF-2α, which in turn drive the transcription of many genes that are important for renal cancer growth and progression, including genes involved in lipid metabolism and signaling. Recent data suggest that HIF-1/2 are themselves regulated by bioactive lipids which make the connection between lipids and renal cancer obvious. This review will summarize the effects and contributions of the different classes of bioactive lipids, including sphingolipids, glycosphingolipids, eicosanoids, free fatty acids, cannabinoids, and cholesterol to renal carcinoma progression. Novel pharmacological strategies interfering with lipid signaling to treat renal cancer will be highlighted.

Potential Pathophysiological Pathways in the Complex Relationships between OSA and Cancer.

Several epidemiological and clinical studies have suggested a relationship between obstructive sleep apnea (OSA) and a higher incidence or severity of cancer. This relationship appears to be dependent on a myriad of factors. These include non-modifiable factors, such as age and gender; and modifiable or preventable factors, such as specific comorbidities (especially obesity), the use of particular treatments, and, above all, the histological type or location of the cancer. Heterogeneity in the relationship between OSA and cancer is also related to the influences of intermittent hypoxemia (a hallmark feature of OSA), among others, on metabolism and the microenvironment of different types of tumoral cells. The hypoxia inducible transcription factor (HIF-1α), a molecule activated and expressed in situations of hypoxemia, seems to be key to enabling a variety of pathophysiological mechanisms that are becoming increasingly better recognized. These mechanisms appear to be operationally involved via alterations in different cellular functions (mainly involving the immune system) and molecular functions, and by inducing modifications in the microbiome. This, in turn, may individually or collectively increase the risk of cancer, which is then, further modulated by the genetic susceptibility of the individual. Here, we provide an updated and brief review of the different pathophysiological pathways that have been identified and could explain the relationship between OSA and cancer. We also identify future challenges that need to be overcome in this intriguing field of research.

MiR-182 Is Upregulated in Prostate Cancer and Contributes to Tumor Progression by Targeting MITF.

Altered expression of microRNA-182-5p (miR-182) has been consistently linked with many cancers, but its specific role in prostate cancer remains unclear. In particular, its contribution to epithelial-to-mesenchymal transition (EMT) in this setting has not been well studied. Therefore, this paper profiles the expression of miR-182 in prostate cancer and investigates how it may contribute to progression of this disease. In vitro experiments on prostate cancer cell lines and in silico analyses of The Cancer Genome Atlas (TCGA) prostate adenocarcinoma (PRAD) datasets were performed. PCR revealed miR-182 expression was significantly increased in prostate cancer cell lines compared to normal prostate cells. Bioinformatic analysis of TCGA PRAD data similarly showed upregulation of miR-182 was significantly associated with prostate cancer and clinical markers of disease progression. Functional enrichment analysis confirmed a significant association of miR-182 and its target genes with EMT. The EMT-linked gene MITF (melanocyte inducing transcription factor) was subsequently shown to be a novel target of miR-182 in prostate cancer cells. Further TCGA analysis suggested miR-182 expression can be an indicator of patient outcomes and disease progression following therapy. In summary, this is the first study to report that miR-182 over-expression in prostate cancer may contribute to EMT by targeting MITF expression. We propose miR-182 as a potentially useful diagnostic and prognostic biomarker for prostate cancer and other malignancies.

Interdependent progression of bidirectional sister replisomes in E. coli.

Bidirectional DNA replication complexes initiated from the same origin remain colocalized in a factory configuration for part or all their lifetimes. However, there is little evidence that sister replisomes are functionally interdependent, and the consequence of factory replication is unknown. Here, we investigated the functional relationship between sister replisomes in Escherichia coli, which naturally exhibits both factory and solitary configurations in the same replication cycle. Using an inducible transcription factor roadblocking system, we found that blocking one replisome caused a significant decrease in overall progression and velocity of the sister replisome. Remarkably, progression was impaired only if the block occurred while sister replisomes were still in a factory configuration - blocking one fork had no significant effect on the other replisome when sister replisomes were physically separate. Disruption of factory replication also led to increased fork stalling and requirement of fork restart mechanisms. These results suggest that physical association between sister replisomes is important for establishing an efficient and uninterrupted replication program. We discuss the implications of our findings on mechanisms of replication factory structure and function, and cellular strategies of replicating problematic DNA such as highly transcribed segments.

KEAP1/NRF2 kao potencijalno ciljno mesto dejstva lekova

Nuclear factor erythroid 2-related factor 2 (NRF2; encoded by NFE2L2) is an inducible transcription factor that regulates the expression of a large network of genes encoding proteins with cytoprotective functions. NRF2 also has a role in the maintenance of mitochondrial and protein homeostasis, and its activation allows adaptation to numerous types of cellular stress. NRF2 is principally regulated at the protein stability level by three main ubiquitin ligase systems, of which the regulation by Kelch-like ECH-associated protein 1 (KEAP1), a substrate adaptor protein for Cul3/Rbx1-based ubiquitin ligase, is best understood. KEAP1 is a multi-functional protein and, in addition to being a substrate adaptor, it is a sensor for electrophiles and oxidants. Pharmacological inactivation of KEAP1 has protective effects in animal models of human disease, and KEAP1 is now widely recognized as a drug target, particularly for chronic diseases, where oxidative stress and inflammation underlie pathogenesis. Many compounds that target KEAP1 have been developed, including electrophiles that bind covalently to cysteine sensors in KEAP1, non-electrophilic protein-protein interaction inhibitors that bind to the Kelch domain of KEAP1, disrupting its interaction with NRF2, and most recently, heterobifunctional proteolysistargeting chimeras (PROTACs) that promote the proteasomal degradation of KEAP1. The drug development of KEAP1-targeting compounds has led to the entry of two compounds, dimethyl fumarate (BG-12, Tecfidera®) and RTA-408 (omaveloxolone, SKYCLARYS®), in clinical practice. In 2013, dimethyl fumarate was licenced as the first oral first-line therapy for relapsingremitting multiple sclerosis and is also used for the treatment of moderate-to-severe plaque psoriasis. In February 2023, omaveloxolone was approved by the United States Food and Drug Administration as the first and only drug for patients with Friedreich's ataxia.

Functions of NO and H2S Signal Molecules Against Plant Abiotic Stress.

Nitric oxide (NO) and hydrogen sulfide (H2S) are two recognized signal molecules in higher plants involved in a wide range of physiological processes and the mechanisms of response against adverse environmental conditions. These molecules can interact to provide an adequate response to palliate the negative impact exerted by stressful conditions, particularly by regulating key components of the metabolism of reactive oxygen species (ROS)to avoid their overproduction and further oxidative damage which, finally, affects cellular functioning. NO and H2S can exert the regulation over the function of susceptible proteins by posttranslational modifications (PTMs) including nitration, S-nitrosation, and persulfidation but also through the regulation of gene expression by the induction of specific transcription factors which modulate the expression of genes encoding proteins related to stress resistance. This chapter encompasses a wide perspective of the signaling and functional relationships between NO and H2S to modulate the overproduction of reactive oxygen species, particularly under abiotic stress conditions.

Disease-associated oligodendrocyte signatures in neurodegenerative disease: the known and unknown.

Disease-associated oligodendrocyte signatures in neurodegenerative disease: the known and unknown.

Protocatechuic Aldehyde Alleviates d -Galactose-Induced Cardiomyocyte Senescence by Regulating the TCF3/ATG5 Axis.

Cardiomyocyte senescence is an independent risk factor for cardiovascular diseases. Protocatechuic aldehyde (PCA) is a natural chemical in the Chinese medicinal herb Salvia miltiorrhiza . PCA could protect against oxidative stress and inflammation in the cardiovascular system. In present study, we treated H9C2 cells with d -galactose to establish an in vitro model of cardiomyocyte senescence and investigated the role and underlying mechanisms of PCA in myocardial cell senescence. It was found that d -galactose induced transcription factor 3 (TCF3) expression and decreased autophagy-related genes 5 (ATG5) expression. Meanwhile, inflammation and senescence were exacerbated by d -galactose. TCF3 transcriptionally inhibited ATG5 expression. TCF3 knockdown abolished the effects of d -galactose on H9C2 by activating ATG5-mediated autophagy. PCA hindered TCF3 and inflammation to alleviate the d -galactose-induced senescence of H9C2 cells in a dose-dependent manner. Whereas, the anti-inflammation and anti-senescence effects of PCA were reversed by TCF3 knockdown. Furthermore, absence of ATG5 partially eliminated the impacts of PCA on H9C2 cells treated with d -galactose. Conclusively, PCA alleviated d -galactose-induced senescence by downregulating TCF3, promoting ATG5-mediated autophagy, and inhibiting inflammation in H9C2 cells. These results elucidated the potential mechanism by which PCA alleviated cardiomyocyte senescence and enabled its application in treating cardiomyocyte senescence.

Aging-associated HELIOS deficiency in naive CD4+ T cells alters chromatin remodeling and promotes effector cell responses.

Immune aging combines cellular defects in adaptive immunity with the activation of pathways causing a low-inflammatory state. Here we examined the influence of age on the kinetic changes in the epigenomic and transcriptional landscape induced by T cell receptor (TCR) stimulation in naive CD4+ T cells. Despite attenuated TCR signaling in older adults, TCR activation accelerated remodeling of the epigenome and induced transcription factor networks favoring effector cell differentiation. We identified increased phosphorylation of STAT5, at least in part due to aberrant IL-2 receptor and lower HELIOS expression, as upstream regulators. Human HELIOS-deficient, naive CD4+ T cells, when transferred into human-synovium-mouse chimeras, infiltrated tissues more efficiently. Inhibition of IL-2 or STAT5 activity in T cell responses of older adults restored the epigenetic response pattern to the one seen in young adults. In summary, reduced HELIOS expression in non-regulatory naive CD4+ T cells in older adults directs T cell fate decisions toward inflammatory effector cells that infiltrate tissue.

DNA methylation landscape of tumor-associated macrophages reveals pathways, transcription factors and prognostic value relevant to triple-negative breast cancer patients.

The accumulation of myeloid cells, particularly tumor-associated macrophages (TAMs), characterizes the tumor microenvironment (TME) of many solid cancers, including breast cancer. Compared to healthy tissue-resident macrophages, TAMs acquire distinct transcriptomes and tumor-promoting functions by largely unknown mechanisms. Here, we hypothesize the involvement of TME signaling and subsequent epigenetic reprogramming of TAMs. Using the 4T1 mouse model of triple-negative breast cancer, we demonstrate that the presence of cancer cells significantly alters the DNA methylation landscape of macrophages and, to a lesser extent, bone marrow-derived monocytes (BMDMs). TAM methylomes, dissected into BMDM-originating and TAM-specific epigenetic programs, implicated transcription factors (TFs) and signaling pathways involved in TAM reprogramming, correlated with cancer-specific gene expression patterns. Utilizing published single-cell gene expression data, we linked microenvironmentally-derived signals to the cancer-specific DNA methylation landscape of TAMs. These integrative analyses highlighted the role of altered cytokine production in the TME (eg, TGF-β, IFN-γ and CSF1) on the induction of specific TFs (eg, FOSL2, STAT1 and RUNX3) responsible for the epigenetic reprogramming of TAMs. DNA methylation deconvolution identified a TAM-specific signature associated with the identified signaling pathways and TFs, corresponding with severe tumor grade and poor prognosis of breast cancer patients. Similarly, immunosuppressive TAM functions were identified, such as induction of the immune inhibitory receptor-ligand PD-L1 by DNA hypomethylation of Cd274. Collectively, these results provide strong evidence that the epigenetic landscapes of macrophages and monocytes are perturbed by the presence of breast cancer, pointing to molecular mechanisms of TAM reprogramming, impacting patient outcomes.

Abstract B027: The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is characterized by epigenetic silencing of differentiation genes and accumulation of aberrant myeloid cells arrested at different stages of myeloid development. The combination of epigenetic and differentiation therapies represents an attractive opportunity for AML patients by promoting chromatin remodeling at differentiation genes and induction of terminal maturation and leukemia debulking. The aim of our work is to define the function of the hypoxia inducible transcription factor HIF2alpha in the pathogenesis of AML. By its genetic inactivation and pharmacological inhibition in AML cell lines and patient-derived xenograft models, we found that HIF2alpha acts as a novel regulator of the AML differentiation block and promotes leukemia progression. Gene expression profiling of HIF2alpha-dependent transcriptome revealed that HIF2alpha mainly promotes the expression of genes involved in transcriptional modulation and epigenetic modifications, whilst inhibiting genes of myeloid maturation and activation. Mechanistically, inhibition of HIF2alpha results in decreased global levels of the heterochromatin marker H3K27me3, which is specifically deposited by the catalytic subunit EZH2 of the Polycomb repressive complex 2. Intriguingly, we found that HIF2alpha and EZH2 cooperate at favoring EZH2-mediated deposition of H3K27me3 at reverse hypoxia responsive elements in the regulatory regions of myeloid maturation genes. Additionally, we demonstrate that HIF2alpha is positively regulated by the pro-differentiation agent all-trans retinoic acid (ATRA), and its inhibition cooperates with ATRA in triggering AML cell differentiation. In conclusion, we provide new mechanistic insights into the role of HIF2alpha in the pathogenesis of AML, by promoting an undifferentiated state via EZH2-mediated epigenetic silencing of myeloid differentiation genes. Importantly, small molecule inhibitors of HIF2alpha have been recently generated and are tested for solid cancers. Therefore, HIF2alpha inhibition may open new therapeutic avenues for AML patients and add therapeutic value to ATRA-based therapies by interrupting HIF2alpha upregulation and promoting an epigenetic state permissive to maturation of leukemic blasts and leukemia exhaustion. Citation Format: Daniela Magliulo, Carolina Caserta, Serena Belluschi, Cristina Fracassi, Kety Giannetti, Raffaella Pini, Ettore Zapparoli, Stefano Beretta, Eleonora Draghi, Marco J. Morelli, Raffaella Di Micco, Bernhard Gentner, Luca Vago, Rosa Bernardi. The transcription factor HIF2alpha partakes in the differentiation block of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B027.

Restoration of Dystrophin Expression in Mdx-Derived Muscle Progenitor Cells Using CRISPR/Cas9 System and Homology-Directed Repair Technology.

Duchenne muscular dystrophy (DMD) is a progressive myopathy caused by mutations in genes encoding dystrophin proteins that ultimately lead to depletion of myogenic progenitor cells (MPCs). Several approaches have been used to correctly express the dystrophin gene in induced pluripotent stem cells (iPSCs), including deletion of mutated exon 23 (ΔEx23) by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated gene 9 (Cas9)-mediated gene editing technology. However, this approach is labor-intensive due to individual colony picking and genotyping to verify allelic modification. Here, we present a protocol to restore the function of the dystrophin gene by using homology-directed repair (HDR)-based CRISPR/Cas9 and inducing myogenic program of reprogrammed iPSCs from Mdx mice by inducible muscle-specific transcription factor MyoD.

E93 promotes transcription of RHG genes to initiate apoptosis during Drosophila salivary gland metamorphosis.

20-hydroxyecdysone (20E) induced transcription factor E93 is important for larval-adult transition, which functions in programmed cell death of larval obsolete tissues, and the formation of adult new tissues. However, the apoptosis-related genes directly regulated by E93 are still ambiguous. In this study, an E93 mutation fly strain was obtained by clustered regularly interspaced palindromic repeats (CRISPR) / CRISPR-associated protein 9-mediated long exon deletion to investigate whether and how E93 induces apoptosis during larval tissues metamorphosis. The transcriptional profile of E93 was consistent with 3 RHG (rpr, hid, and grim) genes and the effector caspase gene drice, and all their expressions peaked at the initiation of apoptosis during the degradation of salivary glands. The transcription expression of 3 RHG genes decreased and apoptosis was blocked in E93 mutation salivary gland during metamorphosis. In contrast, E93 overexpression promoted the transcription of 3 RHG genes, and induced advanced apoptosis in the salivary gland. Moreover, E93 not only enhance the promoter activities of the 3 RHG genes in Drosophila Kc cells in vitro, but also in the salivary gland in vivo. Our results demonstrated that 20E induced E93 promotes the transcription of RHG genes to trigger apoptosis during obsolete tissues degradation at metamorphosis in Drosophila.