Transcription Factors Klf4 Research Articles

Abstract B013: KLF5 drives basal cell identity to promote prostate cancer lineage plasticity

Abstract The purpose of this study was to characterize the phenotypes and drivers of therapy-induced lineage plastic castration resistant prostate cancer (CRPC). Since the majority of primary prostate cancer presents with a luminal cell identity, which relies on androgen receptor (AR) signaling for growth and survival, AR-targeted therapies have become an effective method of slowing progression. However, CRPC cells can develop resistance through reduced or lost dependence on AR. These cells can transition their identity to neuroendocrine CRPC (NEPC), which lacks AR and expresses neuroendocrine lineage specific markers like chromogranin A. More frequently, these cells display transition to a cell identity that lacks AR and neuroendocrine markers and has been termed double-negative CRPC (DNPC). Identifying effective therapeutic targets to treat or prevent DNPC remains a significant clinical challenge. By utilizing prostate cancer cell lines, patient derived xenografts (PDX), and publicly available data from clinical CRPC specimens, we have identified an upregulation in basal cell identity in a subset of CRPC in response to AR-targeted therapies. Additionally, we show that the stem cell transcription factor KLF5, which is highly expressed in healthy prostate basal cells, is uniquely upregulated in CRPC subtypes displaying basal cell identity. In a cell line model of DNPC, we found that knockdown of KLF5 decreased the expression of genes that define basal cell identity. From these results, we conclude that AR-targeted therapies, while effective at targeting luminal-like adenocarcinoma, promote upregulation of KLF5 which can drive a basal cell identity and AR independence. These studies nominate KLF5 signaling as a potential therapeutic target to block the emergence of basal identity, inhibit prostate cancer lineage plasticity, and increase the durability of AR-targeted therapies. Citation Format: Samuel P. Pitzen, Yinjie Qiu, Sarah Munro, Scott M. Dehm. KLF5 drives basal cell identity to promote prostate cancer lineage plasticity [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B013.

Abstract A053: Epigenomic analyses of circulating tumor DNA in patients with metastatic castration resistant prostate cancer (mCRPC) treated with immune checkpoint blockade and PARP inhibition

Abstract Background: Molecular characterization of mCRPC has revealed disruption of DNA damage repair genes that may improve sensitivity to PARP inhibitors such as olaparib (O), which in turn may further complement the activity of the PD-L1 immune checkpoint inhibitor, durvalumab (D), through mechanisms such as activation of the STING pathway. We hypothesized that (epi)genomic analyses of circulating tumor DNA (ctDNA) would identify mechanisms of treatment response in patients with mCRPC treated with O plus D (NCT02484404). Methods: Eligible patients for this study had mCRPC with prior treatment by abiraterone and/or enzalutamide. Study participants were administered 300 mg of O twice daily and 1500 mg of D every 4 weeks. We obtained blood samples in Streck tubes from 38 (out of 60) study participants. Plasma samples were acquired at baseline and at progression. Buffy coat was isolated from each sample. Genomic DNA and cell-free DNA (cfDNA) were separately isolated and assembled into sequencing libraries. Low-pass whole-genome sequencing (3-4 × coverage) was performed for somatic copy number and fragmentomic analyses. In a subset of samples with the greatest cfDNA content, chromatin immunoprecipitation sequencing (ChIP-seq) with antibodies against H3K4me2 and H3K4me3 was performed directly in additional aliquots of plasma. Results: Amongst the 38 patients with at least one cfDNA sample, 5 patients had a radiographic partial response (PR) per RECIST v1.1, 28 patients had stable disease (SD), and 6 patients had progressive disease (PD) during the first two months of treatment. At baseline, ctDNA content in plasma was significantly lower in patients who went on to have PR or SD versus PD (p=0.02). However, upon progression, ctDNA content in plasma was not significantly different between groups. Losses to PTEN and NKX3-1 were significantly enriched in ctDNA from patients with PD (p=0.041 and p=0.047, respectively). By contrast, biallelic combined losses to BRCA2, RB1, BRCA1 and TP53 were observed in all PRs at baseline but not in any patient with PD (p=0.048). In patients with SD, longitudinal fragmentomic analysis inferred that the activity of the mitogenic KLF4 transcription factor (TF) was the most increased upon progression, while activity of the protective HIF2A TF was the most decreased. However, the TF binding motifs predicted by H3K4me2 and H3K4me3 cfDNA ChIP-seq did not significantly change in patients with PD. Conclusions: Distinctive genomic and epigenomic profiles were observed in plasma from patients with responsive vs. progressive disease, and the profiles may predict responses to combination D+O therapy. Deeper whole-genome sequencing of these samples currently in progress will reveal the contribution of germline and somatic point mutations to clinical response. Ongoing efforts to estimate the phylogenetic architecture of patients’ tumors from these plasma samples will enable the inference of drug response phenotypes as a function of evolutionary potential and potentially identify new opportunities to target treatment-resistant disease. Citation Format: Anna Baj, Clara C. Y. Seo, Nicholas T. Terrigino, John R. Bright, S. Thomas Hennigan, Isaiah M. King, Shana Y. Trostel, William D. Figg, William L. Dahut, Jung-Min Lee, David Y. Takeda, Fatima Karzai, Adam G. Sowalsky. Epigenomic analyses of circulating tumor DNA in patients with metastatic castration resistant prostate cancer (mCRPC) treated with immune checkpoint blockade and PARP inhibition [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A053.

KLF5-mediated CDCA5 expression promotes tumor development and progression of epithelial ovarian carcinoma

Cell division cycle associated 5 (CDCA5) is correlated with the development and progression of many malignant tumors. However, little is known about its role in epithelial ovarian cancer (EOC) progression. In this study, the clinical value, biological function and underlying mechanisms of CDCA5 in EOC were evaluated. CDCA5 mRNA and protein levels were substantially upregulated in EOC and had a significant positive correlation with adverse clinicopathological characteristics and a poor prognosis. CDCA5 facilitated proliferation, invasion, and metastasis and disrupted mitochondrial-mediated endogenous apoptosis by activating the cell cycle pathway and inhibiting the P53 pathway in EOC cells. Conversely, knockdown of CDCA5 expression blocked the malignant activities of EOC cells and suppressed the growth of xenograft tumors in vivo. Mechanistically, the transcription factor KLF5 bound to a specific site in the CDCA5 promoter and promoted CDCA5 expression. Moreover, KLF5 overexpression rescued the negative regulation of inhibited CDCA5 expression on EOC cell proliferation. In conclusion, our findings revealed that CDCA5 promoted tumor progression of EOC via the KLF5/CDCA5/cell cycle and P53 axes, which might provide new insights into the roles of CDCA5 in EOC.

N6-methyl-2′-deoxyadenosine promotes self-renewal of BFU-E progenitor in erythropoiesis

Red blood cells supply the oxygen required for all human cells and are in demand for emerging blood-loss therapy. Here we identified N6-methyl-2'-deoxyadenosine (6mdA) as an agonist that promotes the hyperproliferation of burst-forming unit erythroid (BFU-E) progenitor cells. In addition, 6mdA represses the apoptosis of erythroid progenitor cells (EPCs). Combined use of with SCF and EPO enabled cultures of isolated BFU-E to be expanded up to 5,000-fold. Transcriptome analysis showed that 6mdA upregulates the expression of the EPC-associated factors c-Kit, Myb, and Gata2 and downregulates that of the erythroid maturation-related transcription factors Gata1, Spi1, and Klf1. Mechanistic studies suggested that 6mdA enhances and prolongs the activation of erythropoiesis-associated master gene c-Kit and its downstream signaling, leading to expansion and accumulation of EPCs. Collectively, we demonstrate that 6mdA can efficiently stimulate the EPC hyperproliferation and provide a new regenerative medicine recipe to improve exvivo generation of red blood cells.

KLF5 and p53 comprise an incoherent feed-forward loop directing cell-fate decisions following stress

In response to stress, cells make a critical decision to arrest or undergo apoptosis, mediated in large part by the tumor suppressor p53. Yet the mechanisms of these cell fate decisions remain largely unknown, particularly in normal cells. Here, we define an incoherent feed-forward loop in non-transformed human squamous epithelial cells involving p53 and the zinc-finger transcription factor KLF5 that dictates responses to differing levels of cellular stress from UV irradiation or oxidative stress. In normal unstressed human squamous epithelial cells, KLF5 complexes with SIN3A and HDAC2 repress TP53, allowing cells to proliferate. With moderate stress, this complex is disrupted, and TP53 is induced; KLF5 then acts as a molecular switch for p53 function by transactivating AKT1 and AKT3, which direct cells toward survival. By contrast, severe stress results in KLF5 loss, such that AKT1 and AKT3 are not induced, and cells preferentially undergo apoptosis. Thus, in human squamous epithelial cells, KLF5 gates the response to UV or oxidative stress to determine the p53 output of growth arrest or apoptosis.

Abstract 288: Development Of A Phenotypic Screen With 56 Marfan Syndrome Patient-derived Fibroblasts

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the extracellular matrix gene coding for fibrillin-1 (FBN1). MFS leads to aortic aneurysms as key feature. Due to the high genetic diversity within the MFS patient population, genotype-phenotype predictions on disease progression and response to therapy remain challenging. Furthermore, current preclinical murine models represent only a subset of FBN1 mutations, which limits translational studies. In this study, we developed a preclinical model using primary cells of MFS patients that represent genetic and clinical variability in order to develop improved drug strategies. Human primary fibroblasts were isolated from skin biopsies derived from MFS patients (n=56) participating in the COMPARE trial. Human aortic smooth muscle cells (AoSMCs) were isolated from aortic tissue derived from MFS patients undergoing prophylactic aortic surgery (n=4). Long-term cultures in 384-well plates were assessed for the extracellular matrix and disease associated pathways. Analysis of all MFS fibroblasts showed either a reduced or abolished fibrillin-1 extracellular matrix network. Moreover, substantially reduced secretion of fibrillin-1 and asprosin in the culture medium was measured, as compared to healthy control fibroblasts. MFS fibroblasts with an abolished fibrillin-1 matrix were associated with patients with more rapid aortic root diameter growth. Exploring disease-relevant pathways revealed reduced nuclear localization of transcription factor KLF4 in MFS fibroblasts, which associated with the level of fibrillin-1 matrix. As a marker for myofibroblasts, α-smooth muscle actin (ASMA) staining was increased in these MFS fibroblasts. These findings were validated in AoSMC cultures, showing similar MFS fibrillin-1 matrix, nuclear KLF4, and ASMA expression profiles. In conclusion, phenotypic screening of patient-derived cells contributes to improved understanding of disease development and is valuable as preclinical tool for risk assessment and drug development for MFS and related conditions.

Epithelial cells activate fibroblasts to promote esophageal cancer development.

Esophageal squamous-cell carcinoma (ESCC) develops through multistage epithelial cancer formation, i.e., from normal epithelium, low- and high-grade intraepithelial neoplasia to invasive carcinoma. However, how the precancerous lesions progress to carcinoma remains elusive. Here, we report a comprehensive single-cell RNA sequencing and spatial transcriptomic study of 79 multistage esophageal lesions from 29 patients with ESCC. We reveal a gradual and significant loss of ANXA1 expression in epithelial cells due to its transcription factor KLF4 suppression along the lesion progression. We demonstrate that ANXA1 is a ligand to formyl peptide receptor type 2 (FPR2) on fibroblasts that maintain fibroblast homeostasis. Loss of ANXA1 leads to uncontrolled transformation of normal fibroblasts into cancer-associated fibroblasts (CAFs), which can be enhanced by secreted TGF-β from malignant epithelial cells. Given the role of CAFs in cancer, our study underscores ANXA1/FPR2 signaling as an important crosstalk mechanism between epithelial cells and fibroblasts in promoting ESCC.

Klf4 protects thymus integrity during late pregnancy.

Pregnancy causes abrupt thymic atrophy. This atrophy is characterized by a severe decrease in the number of all thymocyte subsets and qualitative (but not quantitative) changes in thymic epithelial cells (TECs). Pregnancy-related thymic involution is triggered by progesterone-induced functional changes affecting mainly cortical TECs (cTECs). Remarkably, this severe involution is rapidly corrected following parturition. We postulated that understanding the mechanisms of pregnancy-related thymic changes could provide novel insights into signaling pathways regulating TEC function. When we analyzed genes whose expression in TECs was modified during late pregnancy, we found a strong enrichment in genes bearing KLF4 transcription factor binding motifs. We, therefore, engineered a Psmb11-iCre : Klf4lox/lox mouse model to study the impact of TEC-specific Klf4 deletion in steady-state conditions and during late pregnancy. Under steady-state conditions, Klf4 deletion had a minimal effect on TEC subsets and did not affect thymic architecture. However, pregnancy-induced thymic involution was much more pronounced in pregnant females lacking Klf4 expression in TECs. These mice displayed a substantial ablation of TECs with a more pronounced loss of thymocytes. Transcriptomic and phenotypic analyses of Klf4 -/- TECs revealed that Klf4 maintains cTEC numbers by supporting cell survival and preventing epithelial-to-mesenchymal plasticity during late pregnancy. We conclude that Klf4 is essential for preserving TEC's integrity and mitigating thymic involution during late pregnancy.

A novel human cellular model of CDA IV enables comprehensive analysis revealing the molecular basis of the disease phenotype

AbstractRed blood cell disorders can result in severe anemia. One such disease congenital dyserythropoietic anemia IV (CDA IV) is caused by the heterozygous mutation E325K in the transcription factor KLF1. However, studying the molecular basis of CDA IV is severely impeded by the paucity of suitable and adequate quantities of material from patients with anemia and the rarity of the disease. We, therefore, took a novel approach, creating a human cellular disease model system for CDA IV that accurately recapitulates the disease phenotype. Next, using comparative proteomics, we reveal extensive distortion of the proteome and a wide range of disordered biological processes in CDA IV erythroid cells. These include downregulated pathways the governing cell cycle, chromatin separation, DNA repair, cytokinesis, membrane trafficking, and global transcription, and upregulated networks governing mitochondrial biogenesis. The diversity of such pathways elucidates the spectrum of phenotypic abnormalities that occur with CDA IV and impairment to erythroid cell development and survival, collectively explaining the CDA IV disease phenotype. The data also reveal far more extensive involvement of KLF1 in previously assigned biological processes, along with novel roles in the regulation of intracellular processes not previously attributed to this transcription factor. Overall, the data demonstrate the power of such a model cellular system to unravel the molecular basis of disease and how studying the effects of a rare mutation can reveal fundamental biology.

Predictive and Regenerative Medicine for Humans

In human history, finding a cure for the devastating disease of cancer has been a long, ongoing battle. Recently, scientific research has shown several feasible bio-medicinal ways for the treatment of cancer. The first method is through predictive regenerative medicine, which converts (breast) cancer cells back into phenotypically benign cells using continually updated predictions from Bayesian inference models. Another common method is the rewriting of damaged coordinating genes, but this has underlying moral issues — who can authorize the right to alter human genes? To investigate these methods, this paper will discuss elementary point-set topologies, physical gene mapping, and gene sequencing. The main purpose of this paper is to establish a statistical method with the help of mathematics in our biological life science. The proposed method can be applied in the case of manipulating genetic transcription and expression with a suitable Bayesian inference model (for genetic reprogramming). The peak value of cancer cell conversion is its Bayesian regression (based on the model’s parameters) from the corresponding Bayesian inference (for the normal posterior distribution, prior and likelihood). The relevant predictive conditions can be determined from the respective model, which could be used to reveal elements that help cancer cell conversion maximization/minimization. Practically, we may apply both of the Bayesian optimization and the related attribute adjustment (to the Oct4, SoX2, Klf4 and c-Myc transcription factors) that can induce the reprogramming of cancer cells. The expected outcome is that we can biomedical-engine the cancer cells conversion. To go a further step, one may apply my proposed Net-Seizing Theory to capture the genetic mutations that underly cancer disease. It is hope that we can design the corresponding universal strategy to heal cancer. Finally, one may establish an intelligent warning mechanism for those high risked cancer candidates but NOT for the thinking control in most of the dictatorship countries.

Modelling the erythroblastic island niche of dyserythropoietic anaemia type IV patients using induced pluripotent stem cells.

Introduction: Congenital dyserythropoietic anaemia (CDA) type IV has been associated with an amino acid substitution, Glu325Lys (E325K), in the transcription factor KLF1. These patients present with a range of symptoms, including the persistence of nucleated red blood cells (RBCs) in the peripheral blood which reflects the known role for KLF1 within the erythroid cell lineage. The final stages of RBCs maturation and enucleation take place within the erythroblastic island (EBI) niche in close association with EBI macrophages. It is not known whether the detrimental effects of the E325K mutation in KLF1 are restricted to the erythroid lineage or whether deficiencies in macrophages associated with their niche also contribute to the disease pathology. Methods: To address this question, we generated an in vitro model of the human EBI niche using induced pluripotent stem cells (iPSCs) derived from one CDA type IV patient as well as two iPSC lines genetically modified to express an KLF1-E325K-ERT2 protein that could be activated with 4OH-tamoxifen. The one patient iPSC line was compared to control lines from two healthy donors and the KLF1-E325K-ERT2 iPSC line to one inducible KLF1-ERT2 line generated from the same parental iPSCS. Results: The CDA patient-derived iPSCs and iPSCs expressing the activated KLF1-E325K-ERT2 protein showed significant deficiencies in the production of erythroid cells with associated disruption of some known KLF1 target genes. Macrophages could be generated from all iPSC lines but when the E325K-ERT2 fusion protein was activated, we noted the generation of a slightly less mature macrophage population marked by CD93. A subtle trend in their reduced ability to support RBC enucleation was also associated with macrophages carrying the E325K-ERT2 transgene. Discussion: Taken together these data support the notion that the clinically significant effects of the KLF1-E325K mutation are primarily associated with deficiencies in the erythroid lineage but it is possible that deficiencies in the niche might have the potential to exacerbate the condition. The strategy we describe provides a powerful approach to assess the effects of other mutations in KLF1 as well as other factors associated with the EBI niche.

Abstract 1484: Identification of the critical active enhancer and regulated transcripts contributing to the refractory nature of pancreatic cancer

Abstract Background: Pancreatic adenocarcinoma (PAAD) is the most lethal human cancer, with a 5-year survival rate of less than 10%. Thus, the identification of prognostic biomarkers as well as molecular therapeutic targets should be critical. Gene expression is regulated by various factors such as transcription factors, promoters, enhancers, DNA methylation, microRNAs, etc. Among them, it has been reported that enhancers play an essential role in the development and progression of cancer. This study aims to identify the novel regulatory element of gene transcription and therapeutic targets in pancreatic cancer by analyzing enhancers. Material and Methods: We implemented NET-CAGE analysis to identify the active enhancer regions in pancreatic cancer cell lines and two immortalized normal pancreatic cell lines. As for the enhancer sequences, we conducted a motif analysis using an analytical pipeline, HOMER to capture sequence motifs enriched in the enhancers. The p-values output by HOMER were applied to identify transcription factor motifs enriched in the tumor cell lines compared to the normal cell lines. We analyzed RNA-sequencing data derived not only from 105 cases of in-house pancreatic cancer but from 178 cases of The Cancer Genome Atlas. Results: We found that the enrichment of KLF and ELF family transcription factors was observed in cancer cell lines compared to normal cell lines. ELF4 expression was significantly higher in tumor tissues than in normal tissues (Wilcoxon rank sum test, p<0.05). In survival analysis, high ELF4 expression was significantly associated with poor prognosis (Kaplan-Meier method, Log-rank test, p<0.05). Immunohistochemical staining showed ELF4 expression in the nuclei of PAAD cells, and GSEA showed a positive correlation between increased ELF4 expression and the cell cycle-related gene sets and PI3K/AKT/mTOR-related gene sets. Conclusions: The current result of the NET-CAGE enhancer analysis revealed that the KLF and ELF families might contribute to activating pancreatic cancer. As we know that KLF4 induces the pluripotency of somatic cells, KLF and ELF are considered to be cancer stemness-related genes. We are focusing on deciphering the regulatory element of the KLF and ELF to orchestrate downstream molecules of cancer stemness-related genes, which will lead to the intractability of pancreatic cancer. Citation Format: Tadashi Abe, Hideyuki Saito, Yusuke Nakano, Masahiro Hashimoto, Yuki Ando, Yuichi Hisamatsu, Takeo Toshima, Yusuke Yonemura, Takaaki Masuda, Ken Eto, Toru Ikegami, Koshi Mimori. Identification of the critical active enhancer and regulated transcripts contributing to the refractory nature of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1484.

Generation of an induced pluripotent stem cell lines NSHDMUi001-A from patients with type 2 diabetes

Type 2 diabetes mellitus (T2DM) is common in China, and its aetiology and pathogenesis are still unclear. We reprogrammed pEP4EO2SEN2K and pEP4EO2SET2K, pCEP4-M2L was electrotransfected in T2DM patients with pEP4EO2SEN2K, and pCEP4-M2L was electrotransfected in T2DM patients expressing the OCT4, SOX2, NANOG, LIN28, c-MYC, KLF4, and SV40LT transcription factors to obtain induced pluripotent stem cells (iPSCs). The obtained iPSCs have been verified to have pluripotency, normal karyotype and differentiation potential; therefore, these cells can be used in the study of disease pathophysiology and drug development to create new therapeutic targets for T2DM and associated central nervous system damage.

Chemokine CCL2 Activates Hypoxia Response Factors Regulating Pluripotency and Directed Endothelial Differentiation of Human Pluripotent Stem Cells

Human pluripotent stem cell (PSC) research is currently focused on selecting conditions and growth factors that better mimic preimplantation development and germ cell differentiation, which is important for disease modeling using PSC. Previously, it was shown that in the presence of the chemokine CCL2, human PSCs acquire properties attributable for preimplantation blastomeres, namely, they activate the JAK-STAT3 signalling pathway and increase the mRNA level of the hypoxic response genes. However, CCL2 is practically not used in the human PSCs cultivation, and its effect is described in a single study. We continued to study the CCL2 effect on human PSC and showed that human embryonic and induced pluripotent stem cells cultured with CCL2 have an increased protein level of the oxygen-dependent subunits HIF1A and HIF2A, which are necessary to trigger the hypoxic response, as well as elevated protein levels of the key pluripotency transcription factors OCT4, NANOG, KLF4, SOX2, and TFCP2L1. In addition, the presence of CCL2 had a positive effect on directed endothelial differentiation, accelerating the maturation of progenitors and enhancing the angiogenic potential of differentiated derivatives.

Case report of schizophrenia in a patient with de novo mutation in the SLC6A1 gene

IntroductionSchizophrenia is a severe mental disorder mainly caused by genetic risk factors. Many studies have demonstrated that both multiple genetic variants and rare mutations are associated with schizophrenia risk. The next step is to study the causal effect of the gene on the phenotype. Recently, a large family-based study identified de novo mutations, which may increase liability to schizophrenia (Rees et al 2020). In particular, a mutation in the GABA transporter (SLC6A1) gene (rs756927822 C/T) was identified in one patient from our subsample.ObjectivesHere, we present a case report of this patient and describe the procedure of derivation of induced pluripotent stem cells (iPSCs) from fibroblast cultures.MethodsClinical, psychometric and neuropsychological methods were used. iPSCs were derived from patients’ and both unaffected parents’ fibroblasts. Human fibroblasts, cultured in fibroblast medium, are infected with lentivirus vectors expressing the transcription factors Oct4, Sox2, c-Myc, and KLF4. All iPSCs were immunocytochemical stained for intracellular (Oct4, Sox2) and extracellular (SSEA4, Tra-1-81, Tra-1-60) pluripotency markers. An qPCR analysis for pluripotency markers (TDGF1, Sox2, Oct4, REX1, LIN28, NANOG, KLF4, GDF3, DPPA4, DNMT3) was performed. All four iPSC lines formed embryoid bodies before the differentiating into three germ layers. Differentiation was confirmed by immunostaining for mesoderm (aSMA), ectoderm (Nestin, Desmin) andendoderm (FoxA2, Pax6) markers.ResultsA 47-year-old male patient was presented to psychiatry at the age of 16. There was no personal or family history of psychiatric disorder, the premorbid functioning was normal, the patient had no somatic diseases, showed high performance in sport (mountain skiing). On his first admission, he was diagnosed with schizoaffective psychosis. The patient showed signs of mania and catatonia. Neuropsychological testing revealed a decrease of cognitive functioning (short-term and associative memory). The patient was followed up for more than 20 years. The diagnosis was changed for schizophrenia at the age of 43 years. There was a deterioration in cognitive function (the apparent decrease in performance on neurocognitive tests (attention, memory, executive functions) from the first examination (1997) till last one (2019). The patient refused or was not able to perform most of the tasks. During follow-up, the patient shows good adherence to treatment.ConclusionsFor this patient, obtained lines might be valuable for investigating the disease mechanisms and screening candidate drugs.Disclosure of InterestNone Declared

Homogeneity of XEN Cells Is Critical for Generation of Chemically Induced Pluripotent Stem Cells.

In induced pluripotent stem cells (iPSCs), pluripotency is induced artificially by introducing the transcription factors Oct4, Sox2, Klf4, and c-Myc. When a transgene is introduced using a viral vector, the transgene may be integrated into the host genome and cause a mutation and cancer. No integration occurs when an episomal vector is used, but this method has a limitation in that remnants of the virus or vector remain in the cell, which limits the use of such iPSCs in therapeutic applications. Chemical reprogramming, which relies on treatment with small-molecule compounds to induce pluripotency, can overcome this problem. In this method, reprogramming is induced according to the gene expression pattern of extra-embryonic endoderm (XEN) cells, which are used as an intermediate stage in pluripotency induction. Therefore, iPSCs can be induced only from established XEN cells. We induced XEN cells using small molecules that modulate a signaling pathway and affect epigenetic modifications, and devised a culture method in which can be produced homogeneous XEN cells. At least 4 passages were required to establish morphologically homogeneous chemically induced XEN (CiXEN) cells, whose properties were similar to those of XEN cells, as revealed through cellular and molecular characterization. Chemically iPSCs derived from CiXEN cells showed characteristics similar to those of mouse embryonic stem cells. Our results show that the homogeneity of CiXEN cells is critical for the efficient induction of pluripotency by chemicals.

MiR579-3p is an inhibitory modulator of neointimal hyperplasia and transcription factors c-MYB and KLF4

Neointimal hyperplasia (IH) is a common vascular pathology that typically manifests in in-stent restenosis and bypass vein graft failure. Smooth muscle cell (SMC) phenotypic switching is central to IH, both regulated by some microRNAs, yet the role of miR579-3p, a scarcely studied microRNA, is not known. Unbiased bioinformatic analysis suggested that miR579-3p was repressed in human primary SMCs treated with different pro-IH cytokines. Moreover, miR579-3p was software-predicted to target both c-MYB and KLF4 − two master transcription factors known to promote SMC phenotypic switching. Interestingly, treating injured rat carotid arteries via local infusion of miR579-3p-expressing lentivirus reduced IH 14 days after injury. In cultured human SMCs, transfection with miR579-3p inhibited SMC phenotypic switching, as indicated by decreased proliferation/migration and increased SMC contractile proteins. miR579-3p transfection downregulated c-MYB and KLF4, and luciferase assays indicated miR579-3p’s targeting of the 3′UTRs of the c-MYB and KLF4 mRNAs. In vivo, immunohistochemistry showed that treatment of injured rat arteries with the miR579-3p lentivirus reduced c-MYB and KLF4 and increased SMC contractile proteins. Thus, this study identifies miR579-3p as a previously unrecognized small-RNA inhibitor of IH and SMC phenotypic switch involving its targeting of c-MYB and KLF4. Further studies on miR579-3p may provide an opportunity for translation to develop IH-mitigating new therapeutics.

Nitazoxanide inhibits acetylated KLF5-induced bone metastasis by modulating KLF5 function in prostate cancer

BackgroundCastration-resistant prostate cancer often metastasizes to the bone, and such bone metastases eventually become resistant to available therapies, leading to the death of patients. Enriched in the bone, TGF-β plays a pivotal role in bone metastasis development. However, directly targeting TGF-β or its receptors has been challenging for the treatment of bone metastasis. We previously found that TGF-β induces and then depends on the acetylation of transcription factor KLF5 at K369 to regulate multiple biological processes, including the induction of EMT, cellular invasiveness, and bone metastasis. Acetylated KLF5 (Ac-KLF5) and its downstream effectors are thus potential therapeutic targets for treating TGF-β-induced bone metastasis in prostate cancer.MethodsA spheroid invasion assay was applied to prostate cancer cells expressing KLF5K369Q, which mimics Ac-KLF5, to screen 1987 FDA-approved drugs for invasion suppression. Luciferase- and KLF5K369Q-expressing cells were injected into nude mice via the tail artery to model bone metastasis. Bioluminescence imaging, micro-CT), and histological analyses were applied to monitor and evaluate bone metastases. RNA-sequencing, bioinformatic, and biochemical analyses were used to understand nitazoxanide (NTZ)-regulated genes, signaling pathways, and the underlying mechanisms. The binding of NTZ to KLF5 proteins was evaluated using fluorescence titration, high-performance liquid chromatography (HPLC), and circular dichroism (CD) analysis.ResultsNTZ, an anthelmintic agent, was identified as a potent invasion inhibitor in the screening and validation assays. In KLF5K369Q-induced bone metastasis, NTZ exerted a potent inhibitory effect in preventive and therapeutic modes. NTZ also inhibited osteoclast differentiation, a cellular process responsible for bone metastasis induced by KLF5K369Q. NTZ attenuated the function of KLF5K369Q in 127 genes’ upregulation and 114 genes’ downregulation. Some genes’ expression changes were significantly associated with worse overall survival in patients with prostate cancer. One such change was the upregulation of MYBL2, which functionally promotes bone metastasis in prostate cancer. Additional analyses demonstrated that NTZ bound to the KLF5 protein, KLF5K369Q bound to the promoter of MYBL2 to activate its transcription, and NTZ attenuated the binding of KLF5K369Q to the MYBL2 promoter.ConclusionsNTZ is a potential therapeutic agent for bone metastasis induced by the TGF-β/Ac-KLF5 signaling axis in prostate cancer and likely other cancers.

The Bovine Herpesvirus 1 Latency-Reactivation Cycle, a Chronic Problem in the Cattle Industry.

Bovine alphaherpesvirus 1 (BoHV-1) is a persistent and recurring disease that affects cattle worldwide. It is a major contributor to bovine respiratory disease and reproductive failure in the US. A major complication of BoHV-1 arises from the lifelong latent infection established in the sensory ganglia of the peripheral nervous system following acute infection. Lifelong latency is marked by periodic reactivation from latency that leads to virus transmission and transient immunosuppression. Physiological and environmental stress, along with hormone fluctuations, can drive virus reactivation from latency, allowing the virus to spread rapidly. This review discusses the mechanisms of the latency/reactivation cycle, with particular emphasis on how different hormones directly regulate BoHV-1 gene expression and productive infection. Glucocorticoids, including the synthetic corticosteroid dexamethasone, are major effectors of the stress response. Stress directly regulates BoHV-1 gene expression through multiple pathways, including β-catenin dependent Wnt signaling, and the glucocorticoid receptor. Related type 1 nuclear hormone receptors, the androgen and progesterone receptors, also drive BoHV-1 gene expression and productive infection. These receptors form feed-forward transcription loops with the stress-induced Krüppel-like transcription factors KLF4 and KLF15. Understanding these molecular pathways is critical for developing novel therapeutics designed to block reactivation and reduce virus spread and disease.

PDC-like cells are pre-DC2 and require KLF4 to control homeostatic CD4 T cells.

Plasmacytoid dendritic cells (pDCs) have been shown to play an important role during immune responses, ranging from initial viral control through the production of type I interferons to antigen presentation. However, recent studies uncovered unexpected heterogeneity among pDCs. We identified a previously uncharacterized immune subset, referred to as pDC-like cells, that not only resembles pDCs but also shares conventional DC (cDC) features. We show that this subset is a circulating precursor distinct from common DC progenitors, with prominent cDC2 potential. Our findings from human CD2-iCre and CD300c-iCre lineage tracing mouse models suggest that a substantial fraction of cDC2s originates from pDC-like cells, which can therefore be referred to as pre-DC2. This precursor subset responds to homeostatic cytokines, such as macrophage colony stimulating factor, by expanding and differentiating into cDC2 that efficiently prime T helper 17 (TH17) cells. Development of pre-DC2 into CX3CR1+ ESAM- cDC2b but not CX3CR1- ESAM+ cDC2a requires the transcription factor KLF4. Last, we show that, under homeostatic conditions, this developmental pathway regulates the immune threshold at barrier sites by controlling the pool of TH17 cells within skin-draining lymph nodes.