Hepatic Leukemia Factor Research Articles

Pan-cancer bioinformatics analysis of hepatic leukemia factor and further validation in colorectal cancer.

Hepatic leukemia factor (HLF) is associated with cancer onset, growth, and progression; however, little is known regarding its biological role in pan-cancer. In order to further evaluate the diagnostic and prognostic value of HLF in pan-cancer and colorectal cancer (CRC), we performed comprehensive bioinformatics analyses of the molecular mechanism of HLF in pan-cancer, with subsequent verification in CRC. We downloaded data (gene expression, clinical data, follow-up duration, and immune-related data) related to 33 solid tumor types from UCSC Xena (University of California Santa Cruz cancer database, https://xena.ucsc.edu/). HLF expression was analyzed in pan-cancer, and its diagnostic efficacy, prognostic value, and correlation with pathological stage and cancer immunity were determined. We also analyzed gene alterations in HLF and biological processes involved in its regulation in pan-cancer. Using CRC data in The Cancer Genome Atlas (TCGA), we assessed correlations between HLF and CRC diagnosis, prognosis, and drug sensitivity and performed functional enrichment analyses. Moreover, we constructed an HLF-related ceRNA regulatory network. Finally, we externally validated HLF expression and diagnostic and prognostic value in CRC using Gene Expression Omnibus (GEO) database, as well as by performing in vitro experiments. HLF expression was downregulated in most tumors, and HLF showed good predictive potential for pan-cancer diagnosis and prognosis. It was closely related to the clinicopathological stages of pan-cancer. Further, HLF was associated with tumor microenvironment and immune cell infiltration in many tumors. Analyses involving cBioPortal revealed changes in HLF amplifications and mutations in most tumors. HLF was also closely associated with microsatellite instability and tumor mutational burden in pan-cancer and involved in regulating various tumor-related pathways and biological processes. In CRC, HLF expression was similarly downregulated, with implications for CRC diagnosis and prognosis. Functional enrichment analysis indicated the association of HLF with many cancer-related pathways. Further, HLF was associated with drug (e.g., oxaliplatin) sensitivity in CRC. The ceRNA regulatory network showed multigene regulation of HLF in CRC. External validation involving GEO databases and quantitative real-time polymerase chain reaction (qRT-PCR) data substantiated these findings. HLF expression generally exhibited downregulation in pan-cancer, contributing to tumor occurrence and development by regulating various biological processes and affecting tumor immune characteristics. HLF was also closely related to CRC occurrence and development. We believe HLF can serve as a reliable diagnostic, prognostic, and immune biomarker for pan-cancer.

Transcriptional regulation of genetic variants in the SLC40A1 promoter.

Solute carrier 40A1 (SLC40A1) encodes ferroportin, which is the only known transmembrane protein that exports elemental iron from mammalian cells and is essential for iron homeostasis. Mutations in SLC40A1 are associated with iron-overload disorders. In addition to ferroportin diseases, SLC40A1 expression is downregulated in various cancer types. Despite the clinical significance of the SLC40A1 transporter, only a few studies have investigated genetic variants in SLC40A1. The present study was performed to identify genetic variations in the SLC40A1 promoter and functionally characterize each variant using in vitro assays. We investigated four haplotypes and five variants in the SLC40A1 promoter. We observed that haplotype 3 (H3) had significantly lower promoter activity than H1, whereas the activity of H4 was significantly higher than that of H1. Luciferase activity of H2 was comparable to that of H1. In addition, four variants of SLC40A1, c.-1355G>C, c.-662C>T, c.-98G>C, and c.-8C>G, showed significantly increased luciferase activity compared to the wild type (WT), whereas c.-750G>A showed significantly decreased luciferase activity compared to the WT. Three transcription factors, cAMP response element-binding protein-1 (CREB-1), chicken ovalbumin upstream promoter transcription factor 1, and hepatic leukemia factor (HLF), were predicted to bind to the promoter regions of SLC40A1 near c.-662C>T, c.-98G>C, and c.-8C>G, respectively. Among these, CREB-1 and HLF bound more strongly to the variant sequences than to the WT and functioned as activators of SLC40A1 transcription. Collectively, our findings indicate that the two SLC40A1 promoter haplotypes affect SLC40A1 transcription, which is regulated by CREB-1 and HLF.

Characterization of Hematopoietic Stem Cell Diversity Using Spatiotemporal Quantitative Phase Imaging

Cells undergo continual transformations, never retaining their original state. Hematopoietic stem cells (HSCs), at the apex of the hematopoietic hierarchy, are not exempt from this dynamic process. In recent years, the advent of single-cell analysis techniques such as scRNAseq has progressively unveiled the heterogeneity within cell populations previously presumed to be homogeneous. However, these techniques are limited to evaluating the cellular state at a single temporal point based on snapshot analysis, thus failing to account for temporal changes and dynamic behavior of cells. Therefore, the true diversity inherent to the ever-changing HSCs remains uncertain. In this study, we comprehensively elucidate HSC diversity, including temporal changes, by integrating our recently established ex vivo HSC expansion techniques (Wilkinson et al., Nature, Becker et al., Cell Stem Cell), single-cell quantitative phase imaging (QPI), and post-transplantation dynamics analysis. For live imaging, HSCs were harvested from hepatic leukemia factor ( Hlf)-tdTomato reporter mice, which specifically marks long-term HSCs. Index sorted single Hlf-tdTomato + HSC were expanded with a single-HSC culture system and monitored by QPI fluorescence time-lapse imaging. Surprisingly, even though the cells were derived from the same HSC fraction, significant variations in mitotic rate, motility, morphological changes, and Hlf expression levels were observed at the single-cell level. Stemness did not decrease stepwise over time, but always exhibited continuous changes. Thus, analysis including the temporal dimension illuminated the infinitely branched nature of HSC diversity. Interestingly, Hlf expression level correlated with the dynamic behavior of HSCs in the expansion culture system. Hlf-high HSCs exhibited a slow division rate, small, rounded morphology and low motility, while Hlf-low HSCs actively divided and differentiated, displayed larger morphological size with low sphericity, and exhibited high motility. Furthermore, spatiotemporal dynamics analysis of Hlf-high/low HSCs after transplantation, employing the highly sensitive in vivo imaging system Akaluc bioluminescence imaging (AkaBLI), revealed that Hlf-high HSCs initiate systemic hematopoiesis slowly, while Hlf-low HSCs expanded quickly, characterized by short-term explosive hematopoiesis in the spleen. As expected, only Hlf-high HSCs had long-term bone marrow reconstitution ability. Thus, based on the expression level of Hlf, it is possible to predict the dynamic behavior of the cells in the expansion medium and in the post-transplant body. Next, we investigated whether Hlf expression level could be predicted based on the kinetic features of cells during HSC ex vivo expansion. Interestingly, multidimensional analysis based on kinetic features extracted from time-lapse imaging showed multiple clusters, each linked to Hlf expression levels. In human HSCs, the dynamic diversity of HSCs was also observed in our recently established ex vivo expansion system (Sakurai et al., Nature). Notably, the kinetic features of human HSCs differed from those of mouse HSCs. Transplantation of human HSCs exhibiting specific morphological characteristics showed long-term reconstitution ability. Thus, classification of HSCs based on kinetic features facilitated enrichment of functional HSCs. This study demonstrates the importance of characterizing cells, including the temporal dimension, and illustrates the existence of infinite dynamic individuality in mouse and human HSCs.

Tensin 1 regulated by hepatic leukemia factor represses the progression of prostate cancer.

Hepatic leukemia factor (HLF), a transcription factor, is dysregulated in many cancers. This study investigates the function of HLF in prostate cancer (PCa) and its relation to tensin 1 (TNS1). Clinical tissues were collected from 24 PCa patients. Duke University 145 (DU145) and PC3 cells overexpressing HLF were established. HLF signaling was downregulated in PCa tissues compared to adjacent tissues and in DU145 and PC3 cells compared to prostate epithelial cells RWPE-1 or prostate stromal cells (WPMY-1). PCa cell lines with overexpression of HLF had reduced proliferative, migratory, and invasive activity, increased apoptosis, and cell mitosis mostly in the G0/G1 phase. HLF induced the TNS1 transcription to activate the p53 pathway. Depletion of TNS1 reversed the anti-tumor effects of HLF on PCa cells and tumor growth and metastasis in vivo. In summary, our findings suggest that HLF suppressed PCa progression by upregulating TNS1 expression and inducing the p53 pathway activation, which might provide insights into novel strategies for combating PCa.

HLF promotes ovarian cancer progression and chemoresistance via regulating Hippo signaling pathway

Hepatic leukemia factor (HLF) is aberrantly expressed in human malignancies. However, the role of HLF in the regulation of ovarian cancer (OC) remains unknown. Herein, we reported that HLF expression was upregulated in OC tissues and ovarian cancer stem cells (CSCs). Functional studies have revealed that HLF regulates OC cell stemness, proliferation, and metastasis. Mechanistically, HLF transcriptionally activated Yes-associated protein 1 (YAP1) expression and subsequently modulated the Hippo signaling pathway. Moreover, we found that miR-520e directly targeted HLF 3′-UTR in OC cells. miR-520e expression was negatively correlated with HLF and YAP1 expression in OC tissues. The combined immunohistochemical (IHC) panels exhibited a better prognostic value for OC patients than any of these components alone. Importantly, the HLF/YAP1 axis determines the response of OC cells to carboplatin treatment and HLF depletion or the YAP1 inhibitor verteporfin abrogated carboplatin resistance. Analysis of patient-derived xenografts (PDXs) further suggested that HLF might predict carboplatin benefits in OC patients. In conclusion, these findings suggest a crucial role of the miR-520e/HLF/YAP1 axis in OC progression and chemoresistance, suggesting potential therapeutic targets for OC.

MiRNA-103-3p-Hlf regulates apoptosis and autophagy by targeting hepatic leukaemia factor in heart failure.

Cardiomyocyte apoptosis is an important factor leading to the occurrence and development of heart failure (HF), which is associated with high mortality of patients with cardiovascular diseases. This study aims to investigate the underlying mechanisms of HF in terms of expression and regulation patterns using bioinformatics and experimental validation. Two HF datasets were collected: a dataset GSE112056 downloaded from the GEO database (including mRNA and miRNA sequencing data) and another is the laboratory-owned mRNA dataset. Differential mRNAs and miRNAs in the two datasets were screened using the raw Bayesian approach method. Gene Ontology was used to perform functional enrichment analysis of the differential mRNAs and co-expression network analysis of the differential mRNAs, combined with nuclear transcription factors in the differential miRNAs and mRNAs for target gene prediction. A HF cell model was constructed using mouse cardiomyocytes (HL-1), and the role and mechanism of miRNA-103-3p-Hlf (hepatic leukaemia factor) in the process of HF was verified by cell transfection, luciferase reporter gene, WB, and qPCR. We found that Hlf gene expression was decreased in the HF model group and strongly correlated with FYCO1 (FYVE and coiled-coil domain-containing protein 1) gene, a phenomenon enriched in apoptotic autophagy-related pathways. MiR-103-3p expression was up-regulated in the HF model group, and its targeting correlation with Hlf was confirmed by luciferase activity assay. In the HL-1 cell model, miR-103-3p significantly promoted apoptosis and inhibited autophagy in HL-1 cells (all P<0.05), and overexpression of the Hlf gene reversed this phenomenon, inhibiting apoptosis and promoting autophagy in HL-1 cells (all P<0.05). MiR-103-3p affects myocardial cells apoptosis and autophagy by targeting Hlf, playing as a potential therapeutic biomarker for HF progression.

M6A RNA methylation-mediated upregulation of HLF promotes intrahepatic cholangiocarcinoma progression by regulating the FZD4/β-catenin signaling pathway

Hepatic leukemia factor (HLF) is aberrantly expressed in human malignancies. However, its role in regulating intrahepatic cholangiocarcinoma (ICC) remains unclear. This study aimed to define the role of HLF in ICC progression. Here, we showed that HLF expression is upregulated in ICC and predicts the poor prognosis in patients. Mechanistically, HLF activation in ICC is mediated by METTL3-dependent m6A methylation of the HLF mRNA. As per the results from the loss- or gain-of-function experiments, HLF promoted the self-renewal, tumorigenicity, proliferation and metastasis of ICC cells. RNA-seq and CUT&Tag analyses showed that frizzled-4 (FZD4) and forkhead box Q1 (FOXQ1) are target genes of HLF. Moreover, FOXQ1 transcriptionally activates METTL3 expression, forming a positive feedback loop, which subsequently activates WNT/β-catenin signaling and downstream tumor stemness. Furthermore, HLF expression was positively correlated with METTL3, IGF2BP3, FZD4 and FOXQ1 expression in ICC tissues in a large ICC cohort. The combined IHC panels exhibited a better prognostic value for patients with ICC than any of these components alone. In conclusions, these findings demonstrated that the METTL3/HLF/FOXQ1 regulatory circuit drove FZD4-mediated WNT/β-catenin activation in ICC progression, suggesting that targeting this axis could be novel therapeutic strategy for ICC.

Abstract PD5-07: PD5-07 Hepatic Leukemia Factor (HLF), a potential therapeutic target in triple negative breast cancer

Abstract The importance of the antitumor immune response in triple negative breast cancer (TNBC) is well established. We showed previously that the extent of tumor-infiltrating lymphocytes (TILs) in the residual disease after neoadjuvant chemotherapy is associated with a better prognosis. By analyzing 99 pretreatment samples we discovered that the expression of the transcription factor Hepatic Leukemia Factor (HLF) in tumor cells is associated with lower infiltration of immune cells, leading to bad prognosis. Thereby, the aim of our project is to investigate the cellular and molecular mechanism regulated by HLF to better understand the tumor immune microenvironment, so we can develop better treatment options for patients with TNBC. Analysis of publicly available datasets of TNBC (METABRIC and TCGA) showed that tumors with low levels of HLF correlate with the upregulation of genes involved in biological processes associated with the regulation of immune response and in particular with neutrophils activation. Analysis of HLF expression patterns in these patients indicate that tumors presenting a mesenchymal profile exhibited higher HLF levels than the other TNBC subtypes. To elucidate the mechanism of this phenomenon regulated by HLF and his implication in anti-tumoral immune response, we developed several human TNBC cell lines knock-out or knock-down for our target gene using CRISPR/Cas9 editing technology. Comparison of the transcriptomes showed that the silencing of HLF in some mesenchymal TNBC cell lines lead to the upregulation of components involved in the attraction and activation of neutrophils, including CXCL8 (Neutrophil-activating peptide 1), a major cytokine directly responsible for the attraction of neutrophils in the tumor site. In vitro migration experiments supported these observations since purified blood circulating neutrophils have defective migration capacity that depends on the presence or absence of HLF. Finally, the co-culture of neutrophils with tumor cells silenced for HLF showed us, using a large panel of antibodies by spectral flow cytometry, an activated state of mature neutrophils. In conclusion, our study suggest that HLF plays a fundamental role in the regulation of genes involved in the control of tumoral immune microenvironment through the attraction and the activation of mature neutrophils. These findings have the potential for important implications since they suggest that HLF can be a potential target in treating TNBC. Citation Format: Ibrahim BOUAKKA, Alicia TRAN-DIEN, Marine SIREROL, Aymeric Silvin, Fabrice Andre, Anna ROCCA. PD5-07 Hepatic Leukemia Factor (HLF), a potential therapeutic target in triple negative breast cancer [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 PD5-07.

Novel pyroptosis-associated genes signature for predicting the prognosis of sarcoma and validation.

Sarcoma is a rare mesenchymal malignant tumor. Recently, pyroptosis has been reported to be a mode of programmed cell death. Nonetheless, levels of pyroptosis-associated genes in sarcoma and its relevance to prognostic outcomes are yet to be elucidated. Sarcoma cases were classified into two subtypes with regards to differentially expressed genes. We established a profile composed of seven genes and classified the sarcoma patients into low- and high-risk groups through least absolute shrinkage and selection operator Cox regression. Survival rate of low-risk sarcoma patients was markedly higher, relative to high-risk group (P<0.001). In combination with clinical features, the risk score was established to be an independent predictive factor for OS of sarcoma patients. Chemotherapeutic drug sensitivity response analysis found 65 drugs with higher drug sensitivity in low-risk, than in high-risk group and 14 drugs with higher drug sensitivity in the high-risk patient group, compared with low-risk patient group. In addition, functional enrichment, pathway and gene mutation of the two modules were analyzed. Finally, we used qRT-PCR to detect the expression of seven pyroptosis-related genes in tumor cells, and human skeletal muscle cells, compared with human skeletal muscle cells, PODXL2, LRRC17, GABRA3, SCUBE3 and RFLNB genes show high expression levels in tumor cells, while IGHG2 and hepatic leukemia factor show low expression levels in tumor cells. Our research suggest that pyroptosis is closely associated with sarcoma, and these findings confirm that pyroptosis-associated seven genes have a critical role in sarcoma and are potential prognostic factors for sarcoma.

Hepatic leukemia factor-expressing paraxial mesoderm cells contribute to the developing brain vasculature.

ABSTRACTRecent genetic lineage tracing studies reveal heterogeneous origins of vascular endothelial cells and pericytes in the developing brain vasculature, despite classical experimental evidence for a mesodermal origin. Here we provide evidence through a genetic lineage tracing experiment that cephalic paraxial mesodermal cells give rise to endothelial cells and pericytes in the developing mouse brain. We show that Hepatic leukemia factor (Hlf) is transiently expressed by cephalic paraxial mesenchyme at embryonic day (E) 8.0-9.0 and the genetically marked E8.0 Hlf-expressing cells mainly contribute to the developing brain vasculature. Interestingly, the genetically marked E10.5 Hlf-expressing cells, which have been previously reported to contain embryonic hematopoietic stem cells, fail to contribute to the vascular cells. Combined, our genetic lineage tracing data demonstrate that a transient expression of Hlf marks a cephalic paraxial mesenchyme contributing to the developing brain vasculature.This article has an associated First Person interview with the first author of the paper.

Independent origins of fetal liver haematopoietic stem and progenitor cells.

Self-renewal and differentiation are tightly controlled to maintain haematopoietic stem cell (HSC) homeostasis in the adult bone marrow1,2. During fetal development, expansion of HSCs (self-renewal) and production of differentiated haematopoietic cells (differentiation) are both required to sustain the haematopoietic system for body growth3,4. However, it remains unclear how these two seemingly opposing tasks are accomplished within the short embryonic period. Here we used in vivo genetic tracing in mice to analyse the formation of HSCs and progenitors from intra-arterial haematopoietic clusters, which contain HSC precursors and express the transcription factor hepatic leukaemia factor (HLF). Through kinetic study, we observed the simultaneous formation of HSCs and defined progenitors-previously regarded as descendants of HSCs5-from the HLF+ precursor population, followed by prompt formation of the hierarchical haematopoietic population structure in the fetal liver in an HSC-independent manner. The transcription factor EVI1 is heterogeneously expressed within the precursor population, with EVI1hi cells being predominantly localized to intra-embryonic arteries and preferentially giving rise to HSCs. By genetically manipulating EVI1 expression, we were able to alter HSC and progenitor output from precursors in vivo. Using fate tracking, we also demonstrated that fetal HSCs are slowly used to produce short-term HSCs at late gestation. These data suggest that fetal HSCs minimally contribute to the generation of progenitors and functional blood cells before birth. Stem cell-independent pathways during development thus offer a rational strategy for the rapid and simultaneous growth of tissues and stem cell pools.

Circadian molecular clock disruption in chronic pulmonary diseases.

The circadian clock is the biochemical oscillator with a near 24-h period that is responsible for generating the circadian rhythms in peripheral organs including the lung. Mounting evidence suggests that circadian clock disruption during chronic lung diseases plays an essential role in augmented oxidative stress, inflammatory response, metabolic imbalances, hypoxia/hyperoxia, mucus secretion, dysregulated autophagy, and alters pulmonary function. Here, we review circadian clock disruption and discuss candidate clock genes that are altered at the transcriptional or translational level in chronic pulmonary diseases. This review aims to provide the current knowledge and understanding of the circadian molecular clock disruption in chronic pulmonary diseases which will further advance the development of novel clock-based therapeutics in the future.

HLF expression defines the human hematopoietic stem cell state

AbstractHematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and can regenerate all blood lineages after transplantation. Despite this clear functional definition, highly enriched isolation of human HSCs can currently only be achieved through combinatorial assessment of multiple surface antigens. Although several transgenic HSC reporter mouse strains have been described, no analogous approach to prospectively isolate human HSCs has been reported. To identify genes with the most selective expression in human HSCs, we profiled population and single-cell transcriptomes of unexpanded and ex vivo cultured cord blood–derived hematopoietic stem and progenitor cells as well as peripheral blood, adult bone marrow, and fetal liver. On the basis of these analyses, we propose the master transcription factor HLF (hepatic leukemia factor) as one of the most specific HSC marker genes. To directly track its expression in human hematopoietic cells, we developed a genomic HLF reporter strategy, capable of selectively labeling the most immature blood cells on the basis of a single engineered parameter. Most importantly, HLF-expressing cells comprise all stem cell activity in culture and in vivo during serial transplantation. Taken together, these results experimentally establish HLF as a defining gene of the human HSC state and outline a new approach to continuously mark these cells with high fidelity.

Identification of Key mRNAs as Prediction Models for Early Metastasis of Pancreatic Cancer Based on LASSO.

Pancreatic cancer is a highly malignant and metastatic tumor of the digestive system. Even after surgical removal of the tumor, most patients are still at risk of metastasis. Therefore, screening for metastatic biomarkers can identify precise therapeutic intervention targets. In this study, we analyzed 96 pancreatic cancer samples from The Cancer Genome Atlas (TCGA) without metastasis or with metastasis after R0 resection. We also retrieved data from metastatic pancreatic cancer cell lines from Gene Expression Omnibus (GEO), as well as collected sequencing data from our own cell lines, BxPC-3 and BxPC-3-M8. Finally, we analyzed the expression of metastasis-related genes in different datasets by the Limma and edgeR packages in R software, and enrichment analysis of differential gene expression was used to gain insight into the mechanism of pancreatic cancer metastasis. Our analysis identified six genes as risk factors for predicting metastatic status by LASSO regression, including zinc finger BED-Type Containing 2 (ZBED2), S100 calcium-binding protein A2 (S100A2), Jagged canonical Notch ligand 1 (JAG1), laminin subunit gamma 2 (LAMC2), transglutaminase 2 (TGM2), and the transcription factor hepatic leukemia factor (HLF). We used these six EMT-related genes to construct a risk-scoring model. The receiver operating characteristic (ROC) curve showed that the risk score could better predict the risk of metastasis. Univariate and multivariate Cox regression analyses revealed that the risk score was also an important predictor of pancreatic cancer. In conclusion, 6-mRNA expression is a potentially valuable method for predicting pancreatic cancer metastasis, assessing clinical outcomes, and facilitating future personalized treatment for patients with ductal adenocarcinoma of the pancreas (PDAC).

High Hepatic leukemia factor expression indicates a favorable survival in glioma patients.

Hepatic leukemia factor (HLF) is an oncogenic transcript factor, but its role in gliomas is unclear.With the open-access data from the Cancer Genome Atlatls (TCGA), HLF expression was compared between normal and glioma tissues and its correlation to patient survival, age, gender, race, and tumor grade was analyzed. Multivariate Cox regression was adopted to explore the independent risk factors for patient survival. Survivals between high and low HLF expression, and high and low model predicted risk subgroups were compared. 1, 2, 3, and 5-year patient survival were predicted with the Cox regression model. Gene set enrichment analysis (GSEA) was performed to predict the potential function of HLF.Expression and clinical data of 5 normal brain samples and 655 glioma samples were obtained from TCGA. HLF expression was downregulated in gliomas than normal brain tissue (P = .007), and negatively related to patient age and advancing tumor grade (P < .001). HLF was a protective factor for patient survival (OR = 0.81, 95%CI 0.67–0.99, P = .035). Patients’ survivals were poorer in low HLF expression subgroups and the Cox regression model predicted high-risk subgroups (P < .001). The accuracy of the model in predicting 1, 2, 3, and 5-year patient survival was 0.864, 0.895, 0.907, and 0.893, respectively. GSEA revealed HLF mainly took part in regulating tumor cell metabolism and cell cycle.HLF was downregulated in gliomas than normal tissue, negatively related to patient age and tumor grade, and was an independent protective factor for glioma patients.

3019 – CRISPR-BASED ENGINEERING OF A REPORTER ALLELE DEMONSTRATES THAT HLF EXPRESSION DEFINES THE HUMAN HAEMATOPOIETIC STEM CELL STATE

Haematopoietic stem cells (HSCs) sustain blood cell homeostasis throughout life and are able to regenerate all blood lineages following transplantation. Despite this clear functional definition, the accurate separation of human HSCs from their differentiated progeny can currently only be achieved through combinatorial measurement of multiple surface antigens. By extensive transcriptomic surveying of bulk and single-cell datasets, we have identified the master transcription factor HLF (Hepatic Leukemia Factor) as the most selective and consistently expressed marker gene in HSC-enriched sub-populations of different ontogenetic sources and ex vivo expanded CD34+ cord blood cells. To directly track its expression, we have engineered a genomic reporter strategy that relies on targeting its 3' non-coding region with a Cas9 ribonucleoprotein complex followed by infection with a recombinant AAV6 vector providing a repair template to introduce a fluorescent gene cassette. This approach routinely achieves high reporter insertion efficiency and is capable of selectively labeling the most immature human blood cells on the basis of a single engineered parameter. Most importantly, use of this reporter demonstrates that HLF-expressing cells comprise all of the stem cell activity in culture and in vivo during serial transplantation. Taken together, our results establish HLF as the most defining gene of the human haematopoietic stem cell state and outline a next-generation approach to mark these cells in real-time with unmatched fidelity.

MicroRNA-98 Inhibits Hepatic Stellate Cell Activation and Attenuates Liver Fibrosis by Regulating HLF Expression.

Liver fibrosis is a major endpoint of patients with chronic liver diseases. The molecular mechanisms behind liver fibrosis remain largely unknown. Many studies have indicated the role of microRNA (miRNA) in hepatic tumorigenesis. But the role of miRNA in liver fibrosis is little known. Activated hepatic stellate cells (HSCs) can secret extracellular matrix proteins (ECM) and are the major contributors to liver fibrosis/cirrhosis. Here, a microarray assay of quiescent and transforming growth factor β1 (TGF-β1) activated HSCs indicated that miR-98 might play a crucial role in liver fibrosis. We found that miR-98 was significantly downregulated in activated HSCs. miR-98 overexpression inhibited HSCs activation. Furthermore, we hypothesized that miR-98 regulated hepatic leukemia factor (HLF) expression by binding to the 3′ UTR of its mRNA directly, as evidenced by luciferase reporter assay. HLF overexpression increased HSCs activation by inducing hypoxia inducible factor-1 alpha (HIF-1α) expression, resulting in the activation of TGF-β/Smad2/3 signaling pathway. Besides, low expression of miR-98 was also found in liver tissues from various fibrotic murine models, including carbon tetrachloride (CCl4), bile duct ligation (BDL), and high-fat diet (HFD)-induced liver fibrosis. miR-98 overexpression in vivo by ago-miR-98 injection could attenuate CCl4-, BDL-, and HFD-induced murine hepatic fibrosis. Meanwhile, miR-98 overexpression suppressed HLF expression and reduced fibrosis marker expression. Collectively, our study demonstrates that miR-98 suppress HSCs activation by targeting HLF directly and interacting with HIF-1α/TGF-β/Smad2/3 signaling pathway, which may be an effective therapeutic target for liver fibrosis.

Downregulation of the circadian rhythm regulator HLF promotes multiple-organ distant metastases in non-small cell lung cancer through PPAR/NF-κb signaling.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death due to its early recurrence and widespread metastatic potential. Accumulating studies have reported that dysregulation of circadian rhythms-associated regulators is implicated in the recurrence and metastasis of NSCLC. Therefore, identification of metastasis-associated circadian rhythm genes is clinically necessary. Here we report that the circadian gene hepatic leukemia factor (HLF), which was dramatically reduced in early-relapsed NSCLC tissues, was significantly correlated with early progression and distant metastasis in NSCLC patients. Upregulating HLF inhibited, while silencing HLF promoted lung colonization, as well as metastasis of NSCLC cells to bone, liver and brain in vivo. Importantly, downexpression of HLF promoted anaerobic metabolism to support anchorage-independent growth of NSCLC cells under low nutritional condition by activating NF-κB/p65 signaling through disrupting translocation of PPARα and PPARγ. Further investigations revealed that both genetic deletion and methylation contribute to downexpression of HLF in NSCLC tissues. In conclusion, our results shed light on a plausible mechanism by which HLF inhibits distant metastasis in NSCLC, suggesting that HLF may serve as a novel target for clinical intervention in NSCLC.

Aberrant expression of PAR bZIP transcription factors is associated with epileptogenesis, focus on hepatic leukemia factor

Epilepsy is a widespread neurological disease characterized by abnormal neuronal activity resulting in recurrent seizures. There is mounting evidence that a circadian system disruption, involving clock genes and their downstream transcriptional regulators, is associated with epilepsy. In this study, we characterized the hippocampal expression of clock genes and PAR bZIP transcription factors (TFs) in a mouse model of temporal lobe epilepsy induced by intrahippocampal injection of kainic acid (KA). The expression of PAR bZIP TFs was significantly altered following KA injection as well as in other rodent models of acquired epilepsy. Although the PAR bZIP TFs are regulated by proinflammatory cytokines in peripheral tissues, we discovered that the regulation of their expression is inflammation-independent in hippocampal tissue and rather mediated by clock genes and hyperexcitability. Furthermore, we report that hepatic leukemia factor (Hlf), a member of PAR bZIP TFs family, is invariably downregulated in animal models of acquired epilepsy, regulates neuronal activity in vitro and its overexpression in dentate gyrus neurons in vivo leads to altered expression of genes associated with seizures and epilepsy. Overall, our study provides further evidence of PAR bZIP TFs involvement in epileptogenesis and points to Hlf as the key player.

Hepatic Leukemia Factor Is Critical for Hematopoietic Stem Cells and Promotes MLL-AF9 Acute Myeloid Leukemia

The transcription factor Hepatic leukemia factor (HLF), initially identified as part of a leukemic fusion protein (E2A-HLF) causing B-lymphoid leukemia in children, is expressed in a wide range of tissues. In the mouse hematopoietic system, HLF is highly expressed in hematopoietic stem cells while being hardly detectable in more restricted progenitor populations. We have previously shown that in adult mice, HLF is essential for preserving the hematopoietic stem cell (HSC) pool during regeneration by maintaining their quiescence (Komorowska et al, Cell reports, 2017). Notably, despite this highly specific expression profile and a critical role in hematopoietic regeneration, mice with double knock-out of HLF alleles (KO) appeared to age normally (18-24 months survival &gt; 90% for both WT and KO). In this study, we investigated the role of HLF in HSC during aging and leukemogenesis. We first confirmed that HLF is expressed all through ontogeny by performing quantitative PCR on HSC and progenitor populations from mouse fetal liver (E14.5 FL) and bone marrow (BM) of aged mice (18-month-old mice). We found that not only is HLF expressed in hematopoietic cells from embryonic to aged mice, but also that the expression profile was highly conserved. In fact, we found that HLF expression decreased during hematopoietic differentiation independently of ontogenic age (HLF/HPRT relative expression: 6 in HSC (LSK-CD150+CD48-), 3 in HSPC (LSK), and &lt;0,2 in CMP (Lin-kit+sca1-CD34+CD16/32-), GMP (Lin-kit+sca1-CD34+CD16/32+), and MEP (Lin-kit+sca1-CD34-CD16/32-). Additionally, hematopoietic parameters as well as lineage distribution in peripheral blood and bone marrow of aged KO mice were within normal range (at the exception of a 50% reduction in platelet blood count, also reported in adult mice). While we found no significant difference in the frequency of immunophenotypic HSC in total BM (LSK-CD34-Flt3-; WT: 0,0006 %, KO: 0,0004 %), aged KO grafts (200 000 total BM cells in 1:1 ratio with competitor) demonstrated a defective capacity to reconstitute primary recipients (donor contribution, WT: 60%, KO: 45 %). In secondary recipients, the defect was further aggravated (donor contribution; WT: 60 %, KO: 20%). Because neither SLAM nor CD34 markers can be used in KO FL, we quantified the percentage of EPCR+ cells within the LSK compartment as a surrogate marker for HSC and found a 2-fold decrease compared to WT controls. In competitive transplantations, KO E14.5 fetal liver cells demonstrated an impaired regenerative capacity in primary (donor contribution; WT: 88.8% (SEM 6.2), KO 63% SEM (16.2); n&gt;5) and secondary recipients (donor contribution: WT: 67% (SEM 2.7), KO: 8.4% (SEM 3.6), n=4). In addition to its function in healthy hematopoiesis, studies have suggested that HLF is a downstream mediator of the MLL/AF9 (MA9) leukemic fusion protein. To address the contribution of HLF to MA9 leukemogenesis, mice were transplanted with either KO- or WT-BM-derived c-Kit+ cells expressing the MA9 fusion protein. Preliminary results showed that HLF did not affect the latency of the leukemia in primary recipients with mice succumbing to a lethal disease at days 40-41 after transplant independently of the leukemic graft genetic background. However, KO leukemic cells had a significantly delayed leukemia development in secondary recipients (time to death; WT: 32-39 days, KO: 39-68 days). Moreover, semi-quantitative PCR showed that not only was HLF expressed in leukemia cells harvested from primary recipients but also that the expression level displayed a tendency to an increase in leukemia cells from secondary recipients. Altogether, our data suggest that while HLF may be dispensable for leukemic transformation, it promotes the development of MLL-AF9 leukemia in mice. We are currently in the process of confirming the role of HLF in MA9 leukemia development. Taken together, we here show that HLF is essential for HSCs from embryonic to aged mice. We further show that HLF is critical for serial propagation of AML cells, suggesting that HLF is needed to maintain leukemia stem cells. Disclosures No relevant conflicts of interest to declare.