Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells.

DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.

Alterations of UHRF family Expression and was regulated by High Risk Type HPV16 in Uterine Cervical Cancer.

The altered protein expression of inverted CCAAT box-binding protein of 90 kDa/ubiquitin-like with PHD and RING finger domains 1 (ICBP90/UHRF1), and Np95-like ring finger protein (NIRF)/UHRF2, which belong to the ubiquitin-like with PHD and RING finger domains (UHRF) family, is linked to tumor malignancy and the progression of various cancers. In this study, we analyzed the UHRF family expression in cervical cancers, and it's regulation by human papillomavirus (HPV). Western blotting was performed to analyze protein expression in cervical cancer cell lines. Immunohistochemical analysis were used to investigate the expression of UHRF family and MIB-1 in cervical cancer tissues. Transfection were done for analyze the relationship between UHRF family and HPVs. We showed that NIRF expression was decreased and ICBP90 expression was increased in cervical cancers compared to normal counterparts. Western blotting also showed that NIRF expression was quite low levels, but ICBP90 was high in human cervical cancer cell lines. Interestingly, ICBP90 was up regulated by high risk type HPV16 E6 and E7, but not low-risk type HPV11. On the other hand, NIRF was down regulated by high risk type HPV16 E6 but not by E7. Low risk type HPV11 E6 did not affect the NIRF expression at all. We propose that ICBP90 overexpression, and reduced NIRF expression, found in cervical cancers, is an important event of a cervical carcinogenesis, and especially ICBP90 may offer a proliferating marker and therapeutic target for treating uterine cervical cancers.

CRISPR-Cas9 screen of E3 ubiquitin ligases identifies TRAF2 and UHRF1 as regulators of HIV latency in primary human T cells.

During HIV infection of CD4+ T cells, ubiquitin pathways are essential to viral replication and host innate immune response; however, the role of specific E3 ubiquitin ligases is not well understood. Proteomics analyses identified 116 single-subunit E3 ubiquitin ligases expressed in activated primary human CD4+ T cells. Using a CRISPR-based arrayed spreading infectivity assay, we systematically knocked out 116 E3s from activated primary CD4+ T cells and infected them with NL4-3 GFP reporter HIV-1. We found 10 E3s significantly positively or negatively affected HIV infection in activated primary CD4+ T cells, including UHRF1 (pro-viral) and TRAF2 (anti-viral). Furthermore, deletion of either TRAF2 or UHRF1 in three JLat models of latency spontaneously increased HIV transcription. To verify this effect, we developed a CRISPR-compatible resting primary human CD4+ T cell model of latency. Using this system, we found that deletion of TRAF2 or UHRF1 initiated latency reactivation and increased virus production from primary human resting CD4+ T cells, suggesting these two E3s represent promising targets for future HIV latency reversal strategies. IMPORTANCE: HIV, the virus that causes AIDS, heavily relies on the machinery of human cells to infect and replicate. Our study focuses on the host cell's ubiquitination system which is crucial for numerous cellular processes. Many pathogens, including HIV, exploit this system to enhance their own replication and survival. E3 proteins are part of the ubiquitination pathway that are useful drug targets for host-directed therapies. We interrogated the 116 E3s found in human immune cells known as CD4+ T cells, since these are the target cells infected by HIV. Using CRISPR, a gene-editing tool, we individually removed each of these enzymes and observed the impact on HIV infection in human CD4+ T cells isolated from healthy donors. We discovered that 10 of the E3 enzymes had a significant effect on HIV infection. Two of them, TRAF2 and UHRF1, modulated HIV activity within the cells and triggered an increased release of HIV from previously dormant or "latent" cells in a new primary T cell assay. This finding could guide strategies to perturb hidden HIV reservoirs, a major hurdle to curing HIV. Our study offers insights into HIV-host interactions, identifies new factors that influence HIV infection in immune cells, and introduces a novel methodology for studying HIV infection and latency in human immune cells.

The dopamine receptor D1 inhibitor, SKF83566, suppresses GBM stemness and invasion through the DRD1-c-Myc-UHRF1 interactions.

BACKGROUND: Extensive local invasion of glioblastoma (GBM) cells within the central nervous system (CNS) is one factor that severely limits current treatments. The aim of this study was to uncover genes involved in the invasion process, which could also serve as therapeutic targets. For the isolation of invasive GBM cells from non-invasive cells, we used a three-dimensional organotypic co-culture system where glioma stem cell (GSC) spheres were confronted with brain organoids (BOs). Using ultra-low input RNA sequencing (ui-RNA Seq), an invasive gene signature was obtained that was exploited in a therapeutic context. METHODS: GFP-labeled tumor cells were sorted from invasive and non-invasive regions within co-cultures. Ui-RNA sequencing analysis was performed to find a gene cluster up-regulated in the invasive compartment. This gene cluster was further analyzed using the Connectivity MAP (CMap) database. This led to the identification of SKF83566, an antagonist of the D1 dopamine receptor (DRD1), as a candidate therapeutic molecule. Knockdown and overexpression experiments were performed to find molecular pathways responsible for the therapeutic effects of SKF83566. Finally, the effects of SKF83566 were validated in orthotopic xenograft models in vivo. RESULTS: Ui-RNA seq analysis of three GSC cell models (P3, BG5 and BG7) yielded a set of 27 differentially expressed genes between invasive and non-invasive cells. Using CMap analysis, SKF83566 was identified as a selective inhibitor targeting both DRD1 and DRD5. In vitro studies demonstrated that SKF83566 inhibited tumor cell proliferation, GSC sphere formation, and invasion. RNA sequencing analysis of SKF83566-treated P3, BG5, BG7, and control cell populations yielded a total of 32 differentially expressed genes, that were predicted to be regulated by c-Myc. Of these, the UHRF1 gene emerged as the most downregulated gene following treatment, and ChIP experiments revealed that c-Myc binds to its promoter region. Finally, SKF83566, or stable DRD1 knockdown, inhibited the growth of orthotopic GSC (BG5) derived xenografts in nude mice. CONCLUSIONS: DRD1 contributes to GBM invasion and progression by regulating c-Myc entry into the nucleus that affects the transcription of the UHRF1 gene. SKF83566 inhibits the transmembrane protein DRD1, and as such represents a candidate small therapeutic molecule for GBMs.

Targeting G9a/DNMT1 methyltransferase activity impedes IGF2-mediated survival in hepatoblastoma.

BACKGROUND: As the variable clinical outcome of patients with hepatoblastoma (HB) cannot be explained by genetics alone, the identification of drugs with the potential to effectively reverse epigenetic alterations is a promising approach to overcome poor therapy response. The gene ubiquitin like with PHD and ring finger domains 1 (UHRF1) represents an encouraging epigenetic target due to its regulatory function in both DNA methylation and histone modifications and its clinical relevance in HB. METHODS: Patient-derived xenograft in vitro and in vivo models were used to study drug response. The mechanistic basis of CM-272 treatment was elucidated using RNA sequencing and western blot experiments. RESULTS: We validated in comprehensive data sets that UHRF1 is highly expressed in HB and associated with poor outcomes. The simultaneous pharmacological targeting of UHRF1-dependent DNA methylation and histone H3 methylation by the dual inhibitor CM-272 identified a selective impact on HB patient-derived xenograft cell viability while leaving healthy fibroblasts unaffected. RNA sequencing revealed downregulation of the IGF2-activated survival pathway as the main mode of action of CM-272 treatment, subsequently leading to loss of proliferation, hindered colony formation capability, reduced spheroid growth, decreased migration potential, and ultimately, induction of apoptosis in HB cells. Importantly, drug response depended on the level of IGF2 expression, and combination assays showed a strong synergistic effect of CM-272 with cisplatin. Preclinical testing of CM-272 in a transplanted patient-derived xenograft model proved its efficacy but also uncovered side effects presumably caused by its strong antitumor effect in IGF2-driven tumors. CONCLUSIONS: The inhibition of UHRF1-associated epigenetic traces, such as IGF2-mediated survival, is an attractive approach to treat high-risk HB, especially when combined with the standard-of-care therapeutic cisplatin.

Circ ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 increases the stability of G9a and ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 messenger RNA through recruiting eukaryotic translation initiation factor 4A3, transcriptionally inhibiting PDZ and homeobox protein domain protein 1, and promotes the metastasis of hepatocellular carcinoma.

BACKGROUND AND AIM: Circular ubiquitin-like, containing PHD and ring finger domains 1 (circUHRF1) is aberrantly upregulated in human hepatocellular carcinoma (HCC) tissues. However, the underlying molecular mechanisms remain obscure. The present study aimed at elucidating the interactive function of circUHRF1-G9a-ubiquitin-like, containing PHD and ring finger domains 1 (UHRF1) mRNA-eukaryotic translation initiation factor 4A3 (EIF4A3)-PDZ and LIM domain 1 (PDLIM1) network in HCC. METHODS: Expression of circUHRF1, mRNAs of G9a, UHRF1, PDLIM1, epithelial-mesenchymal transition (EMT)-related proteins, and Hippo-Yap pathway components was determined by quantitative polymerase chain reaction (Q-PCR), immunofluorescence, or Western blot analysis. Tumorigenic and metastatic capacities of HCC cells were examined by cellular assays including Cell Counting Kit-8, colony formation, wound healing, and transwell assays. Molecular interactions between EIF4A3 and UHRF1 mRNA were detected by RNA pull-down experiment. Complex formation between UHRF1 and PDLIM1 promoter was detected by chromatin immunoprecipitation assay. Co-immunoprecipitation was performed to examine the binding between UHRF1 and G9a. RESULTS: Circular ubiquitin-like, containing PHD and ring finger domains 1, G9a, and UHRF1 were upregulated, while PDLIM1 was downregulated in HCC tissue samples and cell lines. Cellular silencing of circUHRF1 repressed HCC proliferation, invasion, migration, and EMT. G9a formed a complex with UHRF1 and inhibited PDLIM1 transcription. CONCLUSION: Eukaryotic translation initiation factor 4A3 regulated circUHRF1 expression by binding to UHRF1 mRNA promoter. circUHRF1 increased the stability of G9a and UHRF1 mRNAs through recruiting EIF4A3. Overexpression of circUHRF1 aggravated HCC progression through Hippo-Yap pathway and PDLIM1 inhibition. By elucidating the molecular function of circUHRF1-G9a-UHRF1 mRNA-EIF4A3-PDLIM1 network, our data shed light on the HCC pathogenesis and suggest a novel therapeutic strategy for future HCC treatment.

Insight into the mechanism of AML del(9q) progression: hnRNP K targets the myeloid master regulators CEBPA (C/EBPα) and SPI1 (PU.1).

Deletions on the long arm of chromosome 9 (del(9q)) are recurrent abnormalities in about 2 % of acute myeloid leukemia cases, which usually involve HNRNPK and are frequently associated with other known aberrations. Based on an Hnrnpk haploinsufficient mouse model, a recent study demonstrated a function of hnRNP K in pathogenesis of myeloid malignancies via the regulation of cellular proliferation and myeloid differentiation programs. Here, we provide evidence that reduced hnRNP K expression results in the dysregulated expression of C/EBPα and additional transcription factors. CyTOF analysis revealed monocytic skewing with increased levels of mature myeloid cells. To explore the role of hnRNP K during normal and pathological myeloid differentiation in humans, we characterized hnRNP K-interacting RNAs in human AML cell lines. Notably, RNA-sequencing revealed several mRNAs encoding key transcription factors involved in the regulation of myeloid differentiation as targets of hnRNP K. We showed that specific sequence motifs confer the interaction of SPI1 and CEBPA 5' and 3'UTRs with hnRNP K. The siRNA mediated reduction of hnRNP K in human AML cells resulted in an increase of PU.1 and C/EBPα that is most pronounced for the p30 isoform. The combinatorial treatment with the inducer of myeloid differentiation valproic acid resulted in increased C/EBPα expression and myeloid differentiation. Together, our results indicate that hnRNP K post-transcriptionally regulates the expression of myeloid master transcription factors. These novel findings can inaugurate novel options for targeted treatment of AML del(9q) by modulation of hnRNP K function.

Enhanced Transcriptional Signature and Expression of Histone-Modifying Enzymes in Salivary Gland Tumors.

Salivary gland tumors (SGTs) are rare and complex neoplasms characterized by heterogenous histology and clinical behavior as well as resistance to systemic therapy. Tumor etiology is currently under elucidation and an interplay of genetic and epigenetic changes has been proposed to contribute to tumor development. In this work, we investigated epigenetic regulators and histone-modifying factors that may alter gene expression and participate in the pathogenesis of SGT neoplasms. We performed a detailed bioinformatic analysis on a publicly available RNA-seq dataset of 94 ACC tissues supplemented with clinical data and respective controls and generated a protein-protein interaction (PPI) network of chromatin and histone modification factors. A significant upregulation of TP53 and histone-modifying enzymes SUV39H1, EZH2, PRMT1, HDAC8, and KDM5B, along with the upregulation of DNA methyltransferase DNMT3A and ubiquitin ligase UHRF1 mRNA levels, as well as a downregulation of lysine acetyltransferase KAT2B levels, were detected in ACC tissues. The protein expression of p53, SUV39H1, EZH2, and HDAC8 was further validated in SGT tissues along with their functional deposition of the repressive histone marks H3K9me3 and H3K27me3, respectively. Overall, this study is the first to detect a network of interacting proteins affecting chromatin structure and histone modifications in salivary gland tumor cells, further providing mechanistic insights in the molecular profile of SGTs that confer to altered gene expression programs.

TET2 lesions enhance the aggressiveness of CEBPA-mutant acute myeloid leukemia by rebalancing GATA2 expression.

The myeloid transcription factor CEBPA is recurrently biallelically mutated (i.e., double mutated; CEBPADM) in acute myeloid leukemia (AML) with a combination of hypermorphic N-terminal mutations (CEBPANT), promoting expression of the leukemia-associated p30 isoform, and amorphic C-terminal mutations. The most frequently co-mutated genes in CEBPADM AML are GATA2 and TET2, however the molecular mechanisms underlying this co-mutational spectrum are incomplete. By combining transcriptomic and epigenomic analyses of CEBPA-TET2 co-mutated patients with models thereof, we identify GATA2 as a conserved target of the CEBPA-TET2 mutational axis, providing a rationale for the mutational spectra in CEBPADM AML. Elevated CEBPA levels, driven by CEBPANT, mediate recruitment of TET2 to the Gata2 distal hematopoietic enhancer thereby increasing Gata2 expression. Concurrent loss of TET2 in CEBPADM AML induces a competitive advantage by increasing Gata2 promoter methylation, thereby rebalancing GATA2 levels. Of clinical relevance, demethylating treatment of Cebpa-Tet2 co-mutated AML restores Gata2 levels and prolongs disease latency.

[Prognostic significance and immune cell infiltration analysis of differentially expressed genes in malignant pleural mesothelioma].

Objective: To explore and analyze differential expressed genes in malignant pleural mesothelioma (MPM) by bioinformatics method, and to study their prognostic value in MPM and their potential role in immunotherapy. Methods: In January 2022, the dataset GSE51024 was downloaded from the GEO database, and MPM (55 cases) and normal tissue (41 cases) samples were obtained. Using R software and HMDD and miRNet database, MPM-related differential genes were screened and co-expressed genes were identified. Co-expressed genes were enriched and functionally annotated, and protein-protein interaction (PPI) networks were constructed and key genes were identified using the STRING database and Cytoscape software. TRRUST and GEPIA databases were used to predict transcription factors of key genes and to analyze prognosis and survival. The correlation between key genes and the degree of infiltration of immune cells was analyzed using TIMER. Results: A total of 435 co-expressed genes were obtained, which were mainly concentrated in the extracellular matrix tissue and the signaling pathways of cell adhesion molecules. Combined with PPI and TRRUST database, seven key MPM prognostic genes were identified. Among them, cyclin 20 (CDC20) , cell cycle checkpoint kinase 1 (CHEK1) , enhancer of Zeste homolog 2 (EZH2) , ribonucleotide reductase subunit M2 (RRM2) , topoisomerase 2A (TOP2A) , ubiquitin like plant homeodomain and ring finger domain 1 (UHRF1) were significantly up-regulated in MPM, while cyclin A1 (CCNA1) was significantly down-regulated. The expressions of CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 genes were significantly associated with MPM overall survival (P<0.05) . The expressions of CDC20, CHEK1, EZH2, RRM2 and TOP2A genes were positively correlated with B cells and dendritic cells (P<0.05) , and negatively correlated with neutrophils (P<0.05) . Conclusion: CCNA1, CDC20, CHEK1, EZH2, RRM2, TOP2A and UHRF1 may be potential prognostic markers in MPM patients, and their expressions may be related to MPM tumor immunity.

Downregulation of DNA methylation enhances differentiation of THP-1 cells and induces M1 polarization of differentiated macrophages.

DNA methylation is an epigenetic modification that regulates gene expression and plays an essential role in hematopoiesis. UHRF1 and DNMT1 are both crucial for regulating genome-wide maintenance of DNA methylation. Specifically, it is well known that hypermethylation is crucial characteristic of acute myeloid leukemia (AML). However, the mechanism underlying how DNA methylation regulates the differentiation of AML cells, including THP-1 is not fully elucidated. In this study, we report that UHRF1 or DNMT1 depletion enhances the phorbol-12-myristate-13-acetate (PMA)-induced differentiation of THP-1 cells. Transcriptome analysis and genome-wide methylation array results showed that depleting UHRF1 or DNMT1 induced changes that made THP-1 cells highly sensitive to PMA. Furthermore, knockdown of UHRF1 or DNMT1 impeded solid tumor formation in xenograft mouse model. These findings suggest that UHRF1 and DNMT1 play a pivotal role in regulating differentiation and proliferation of THP-1 cells and targeting these proteins may improve the efficiency of differentiation therapy in AML patients.

Inhibition of abnormal C/EBPß/α-Syn signaling pathway through activation of Nrf2 ameliorates Parkinson's disease-like pathology.

Parkinson's disease (PD) is characterized by the formation of Lewy bodies (LBs) in the brain. These LBs are primarily composed of α-Synuclein (α-Syn), which has aggregated. A recent report proposes that CCAAT/enhancer-binding proteins ß (C/EBPß) may act as an age-dependent transcription factor for α-Syn, thereby initiating PD pathologies by regulating its transcription. Potential therapeutic approaches to address PD could involve targeting the regulation of α-Syn by C/EBPß. This study has revealed that Nrf2, also known as nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), suppresses the transcription of C/EBPß in SH-SY5Y cells when treated with MPP+ . To activate Nrf2, sulforaphane, an Nrf2 activator, was administered. Additionally, C/EBPß was silenced using C/EBPß-DNA/RNA heteroduplex oligonucleotide (HDO). Both approaches successfully reduced abnormal α-Syn expression in primary neurons treated with MPP+ . Furthermore, sustained activation of Nrf2 via its activator or inhibition of C/EBPß using C/EBPß-HDO resulted in a reduction of aberrant α-Syn expression, thus leading to an improvement in the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc) in mouse models induced by 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) and those treated with preformed fibrils (PFFs). The data presented in this study illustrate that the activation of Nrf2 may provide a potential therapeutic strategy for PD by inhibiting the abnormal C/EBPß/α-Syn signaling pathway.

UHRF1 is a mediator of KRAS driven oncogenesis in lung adenocarcinoma.

KRAS is a frequent driver in lung cancer. To identify KRAS-specific vulnerabilities in lung cancer, we performed RNAi screens in primary spheroids derived from a Kras mutant mouse lung cancer model and discovered an epigenetic regulator Ubiquitin-like containing PHD and RING finger domains 1 (UHRF1). In human lung cancer models UHRF1 knock-out selectively impaired growth and induced apoptosis only in KRAS mutant cells. Genome-wide methylation and gene expression analysis of UHRF1-depleted KRAS mutant cells revealed global DNA hypomethylation leading to upregulation of tumor suppressor genes (TSGs). A focused CRISPR/Cas9 screen validated several of these TSGs as mediators of UHRF1-driven tumorigenesis. In vivo, UHRF1 knock-out inhibited tumor growth of KRAS-driven mouse lung cancer models. Finally, in lung cancer patients high UHRF1 expression is anti-correlated with TSG expression and predicts worse outcomes for patients with KRAS mutant tumors. These results nominate UHRF1 as a KRAS-specific vulnerability and potential target for therapeutic intervention.

The transcription factor HBP1 promotes ferroptosis in tumor cells by regulating the UHRF1-CDO1 axis.

The induction of ferroptosis in tumor cells is one of the most important mechanisms by which tumor progression can be inhibited; however, the specific regulatory mechanism underlying ferroptosis remains unclear. In this study, we found that transcription factor HBP1 has a novel function of reducing the antioxidant capacity of tumor cells. We investigated the important role of HBP1 in ferroptosis. HBP1 down-regulates the protein levels of UHRF1 by inhibiting the expression of the UHRF1 gene at the transcriptional level. Reduced levels of UHRF1 have been shown to regulate the ferroptosis-related gene CDO1 by epigenetic mechanisms, thus up-regulating the level of CDO1 and increasing the sensitivity of hepatocellular carcinoma and cervical cancer cells to ferroptosis. On this basis, we constructed metal-polyphenol-network coated HBP1 nanoparticles by combining biological and nanotechnological. MPN-HBP1 nanoparticles entered tumor cells efficiently and innocuously, induced ferroptosis, and inhibited the malignant proliferation of tumors by regulating the HBP1-UHRF1-CDO1 axis. This study provides a new perspective for further research on the regulatory mechanism underlying ferroptosis and its potential role in tumor therapy.

A novel deformable liposomal hydrogel loaded with a SREBP-1-inhibiting polypeptide for reducing sebum synthesis in golden hamster model.

Excessive sebum is the major factor involved in the pathophysiology of seborrheic diseases. Chemical medicines can result in mild to severe side effects. Polypeptides with much less side effects make them ideal for reducing sebum synthesis. Sterol regulatory element-binding proteins-1 (SREBP-1) is necessary for the biosynthesis of sterols. A SREBP-1-inhibiting polypeptide (SREi), which competitively inhibits the ubiquitination of Insig-1 so as to suppress the activation of SREBP-1 was selected as an active ingredient and formulated into skin topical preparations. The SREi anionic deformable liposomes contained sodium deoxycholate (SDCh) at the concentration of 4.4 mg/mL (SREi-ADL3) and SREi-ADL3 in 0.3% (w/v) carbomer hydrogel (SREi-ADL3-GEL) were prepared and characterized. The SREi-ADL3 presented a high entrapment efficiency of 92.62 ± 6.32%, a particle size of 99.54 ± 7.56 nm and a surface charge of -19.18 ± 0.45 mV. SREi-ADL3-GEL exhibited a sustained release behavior, a higher stability, a much more cellular uptake ability and transdermal absorption. In vivo golden hamster model confirmed that SREi-ADL3-GEL presented the strongest inhibitory effect on sebaceous gland growth and sebum synthesis by down-regulating the mRNA and protein expression of SREBP-1, fatty acid synthase (FAS) and acetyl-coenzyme A carboxylase 1 (ACC1). As confirmed by histological analysis, only a small amount of sebaceous gland lobes with the lightest staining intensity and the smallest dyeing area could be observed in the SREi-ADL3-GEL group. Taken together, SREi-ADL3-GEL displayed potential applications in sebum excessive production related diseases.

Sporadic and Familial Acute Myeloid Leukemia with CEBPA Mutations.

PURPOSE OF REVIEW: CCAAT enhancer binding protein A (CEBPA) gene mutation is one of the common genetic alterations in acute myeloid leukemia (AML), which can be associated with sporadic and familial AML. RECENT FINDINGS: Due to the recent advances in molecular testing and the prognostic role of CEBPA mutation in AML, the definition for AML with CEBPA mutation (AML-CEBPA) has significantly changed. This review provides the rationale for the updates on classifications, and the impacts on laboratory evaluation and clinical management for sporadic and familial AML-CEBPA patients. In addition, minimal residual disease assessment post therapy to stratify disease risk and stem cell transplant in selected AML-CEBPA patients are discussed. Taken together, the recent progresses have shifted the definition, identification, and management of patients with AML-CEBPA.

Alterations of UHRF Family Expression and UHRF1/ICBP90 Inhibits Phosphatase and Tensin Homolog Expression in Endometrial Cancer.

INTRODUCTION: The altered protein expression of inverted CCAAT box-binding protein of 90 kDa/ubiquitin-like with PHD and RING finger domains 1 (ICBP90/UHRF1) and Np95-like ring finger protein (NIRF)/UHRF2, which belong to the ubiquitin-like with PHD and RING finger domains (UHRF) family, is linked to tumor malignancy and the progression of various cancers. To determine the role of NIRF and ICBP90 in endometrial tumorigenesis, we evaluated ICBP90 and NIRF expression levels in endometrial cancers. Also molecular alterations of phosphatase and tensin homolog (PTEN) expression are the important event for endometrial carcinogenesis; therefore, we investigated the involvement between ICBP90 and PTEN expression. METHODS: We used Western blot for NIRF, ICBP90, and PTEN expression, mutation analysis of NIRF gene, and immunohistochemical staining for the expression of NIRF and ICBP90. For immunohistochemical staining, we examined atypical endometrial hyperplasia, endometrial cancers, and noncancerous samples. RESULTS: Our data showed that the reduced expression of NIRF and overexpression of ICBP90 occurred in atypical endometrial hyperplasia and endometrial cancer compared to the normal endometrium. The decrease in NIRF expression was significantly correlated with histological grade. Expression of ICBP90 was high, especially in the peripheral margin of a cancer nest. Western blot analysis of endometrial cancer cell lines referred an opposite correlation between ICBP90 and PTEN expression. CONCLUSION: Our findings suggested that continually overexpressed ICBP90 may contribute to the inhibition of PTEN expression, which is a frequent and important event in endometrial carcinogenesis. We propose that the reduced NIRF expression and ICBP90 overexpression is an early event in endometrial carcinogenesis; thus ICBP90 may be useful as a therapeutic target in this disease.

Inhibitors of UHRF1 base flipping activity showing cytotoxicity against cancer cells.

Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is a nuclear multi-domain protein overexpressed in numerous human cancer types. We previously disclosed the anthraquinone derivative UM63 that inhibits UHRF1-SRA domain base-flipping activity, although having DNA intercalating properties. Herein, based on the UM63 structure, new UHRF1-SRA inhibitors were identified through a multidisciplinary approach, combining molecular modelling, biophysical assays, molecular and cell biology experiments. We identified AMSA2 and MPB7, that inhibit UHRF1-SRA mediated base flipping at low micromolar concentrations, but do not intercalate into DNA, which is a key advantage over UM63. These molecules prevent UHRF1/DNMT1 interaction at replication forks and decrease the overall DNA methylation in cells. Moreover, both compounds specifically induce cell death in numerous cancer cell lines, displaying marginal effect on non-cancer cells, as they preferentially affect cells with high level of UHRF1. Overall, these two compounds are promising leads for the development of anti-cancer drugs targeting UHRF1.

PIP4K2B is mechanoresponsive and controls heterochromatin-driven nuclear softening through UHRF1.

Phosphatidylinositol-5-phosphate (PtdIns5P)-4-kinases (PIP4Ks) are stress-regulated phosphoinositide kinases able to phosphorylate PtdIns5P to PtdIns(4,5)P2. In cancer patients their expression is typically associated with bad prognosis. Among the three PIP4K isoforms expressed in mammalian cells, PIP4K2B is the one with more prominent nuclear localisation. Here, we unveil the role of PIP4K2B as a mechanoresponsive enzyme. PIP4K2B protein level strongly decreases in cells growing on soft substrates. Its direct silencing or pharmacological inhibition, mimicking cell response to softness, triggers a concomitant reduction of the epigenetic regulator UHRF1 and induces changes in nuclear polarity, nuclear envelope tension and chromatin compaction. This substantial rewiring of the nucleus mechanical state drives YAP cytoplasmic retention and impairment of its activity as transcriptional regulator, finally leading to defects in cell spreading and motility. Since YAP signalling is essential for initiation and growth of human malignancies, our data suggest that potential therapeutic approaches targeting PIP4K2B could be beneficial in the control of the altered mechanical properties of cancer cells.

Lenvatinib inhibited HCC cell migration and invasion through regulating the transcription and ubiquitination of UHRF1 and DNMT1.

Hepatocellular carcinoma (HCC) is one of the most common causes of malignancy-related deaths. Lenvatinib, as a multi-targeted tyrosine kinase inhibitor, has gained increasing attention for its antitumor activity. However, the effect and mechanisms of Lenvatinib on HCC metastasis are virtually unknown. In this study, we revealed that Lenvatinib inhibited HCC cell motility and epithelial mesenchymal transition (EMT), along with cell adhesion and extension. Concomitant high DNMT1 and UHRF1 mRNA levels were in HCC patients and indicated worse prognosis. On the one hand, Lenvatinib modulated the transcription of UHRF1 and DNMT1via negatively regulation of ERK/MAPK pathway. On the other hand, Lenvatinib downregulated DNMT1 and UHRF1 expression by promoting their protein degradation through ubiquitin-proteasome pathway, consequently, resulting in upregulation of E-Cadherin. Moreover, Lenvatinib attenuated Huh7 cell adhesion and metastasis in vivo. Our findings provided insight into the intriguing molecular mechanisms regarding the anti-metastasis effect of Lenvatinib in HCC.