E2F Target Genes Research Articles

N6-methyladenosine-induced SVIL antisense RNA 1 restrains lung adenocarcinoma cell proliferation by destabilizing E2F1

ABSTRACT Accumulating evidence indicates that N6-methyladenosine (m6A) and long noncoding RNAs (lncRNAs) play crucial roles in cancer development. However, the biological roles of m6A and lncRNAs in lung cancer tumorigenesis are largely unknown. In this study, SVIL antisense RNA 1 (SVIL-AS1) was downregulated in lung adenocarcinoma (LUAD) tissues and was associated with a favorable prognosis in patients with LUAD. SVIL-AS1 overexpression suppressed LUAD cell proliferation and blocked cell cycle arrest. Mechanistically, METTL3 increased the m6A modification and transcript stability of SVIL-AS1. The enhanced SVIL-AS1 expression mediated by METTL3 suppressed E2F1 and E2F1-target genes. Moreover, SVIL-AS1 accelerated E2F1 degradation. The reduction in cell proliferation induced by SVIL-AS1 overexpression could be rescued by E2F1 overexpression or METTL3 knockdown. In conclusion, our work demonstrated the role and mechanism of METTL3-induced SVIL-AS1 in LUAD, which connects m6A and lncRNA in lung cancer carcinogenesis.

TRNA biogenesis and specific aminoacyl-tRNA synthetases regulate senescence stability under the control of mTOR.

Oncogenes or chemotherapy treatments trigger the induction of suppressive pathways such as apoptosis or senescence. Senescence was initially defined as a definitive arrest of cell proliferation but recent results have shown that this mechanism is also associated with cancer progression and chemotherapy resistance. Senescence is therefore much more heterogeneous than initially thought. How this response varies is not really understood, it has been proposed that its outcome relies on the secretome of senescent cells and on the maintenance of their epigenetic marks. Using experimental models of senescence escape, we now described that the stability of this proliferative arrest relies on specific tRNAs and aminoacyl-tRNA synthetases. Following chemotherapy treatment, the DNA binding of the type III RNA polymerase was reduced to prevent tRNA transcription and induce a complete cell cycle arrest. By contrast, during senescence escape, specific tRNAs such as tRNA-Leu-CAA and tRNA-Tyr-GTA were up-regulated. Reducing tRNA transcription appears necessary to control the strength of senescence since RNA pol III inhibition through BRF1 depletion maintained senescence and blocked the generation of escaping cells. mTOR inhibition also prevented chemotherapy-induced senescence escape in association with a reduction of tRNA-Leu-CAA and tRNA-Tyr-GTA expression. Further confirming the role of the tRNA-Leu-CAA and tRNA-Tyr-GTA, results showed that their corresponding tRNA ligases, LARS and YARS, were necessary for senescence escape. This effect was specific since the CARS ligase had no effect on persistence. By contrast, the down-regulation of LARS and YARS reduced the emergence of persistent cells and this was associated with the modulation of E2F1 target genes expression. Overall, these findings highlight a new regulation of tRNA biology during senescence and suggest that specific tRNAs and ligases contribute to the strength and heterogeneity of this tumor suppressive pathway.

Genome-wide CRISPR/Cas9 knockout screening uncovers ZNF319 as a novel tumor suppressor critical for breast cancer metastasis

Breast cancer is prone to relapse and metastasize to many vital organs, contributing to most of the breast cancer-related death and accentuating the importance of systematic identification of key factors regulating the metastasis of breast cancer. In this study, we performed a genome-wide CRISPR/Cas9 knock out screen in an orthotopic murine model of breast cancer for essential genes monitoring the progression and metastasis of breast cancer. We found one member of the zinc finger protein (ZNF) family, i.e., ZNF319, was among the top candidate genes. We further confirmed the lower expression of ZNF319 in the tumor tissue of breast cancer patients by analyzing tissue sections with IHC staining and TCGA database. Consistently, higher expression of ZNF319 correlates with better clinical outcome in almost all subtypes of breast cancer. Moreover, knocking down or overexpressing ZNF319 in breast cancer cells dramatically affects the breast cancer growth and metastasis capacity both in vitro and in vivo, suggesting ZNF319 functions as a strong suppressor of breast cancer progression. Lastly, the transcriptome analysis on ZNF319-silenced breast cancer cells shows that ZNF319 is involved in multiple crucial signaling pathways and biological processes, especially in cell cycle and proliferation. GO and KEGG analyses of our RNA-seq results reveal the up-regulation of E2F and G2/M related genes in ZNF319-silenced cells, suggesting that ZNF319 monitors the cell cycle during the breast cancer progression through the regulation of the E2F target genes and G2/M checkpoint. In summary, our study identifies ZNF319 as a novel metastasis suppressor gene arresting tumor cell cycle in breast cancer and thus presents a novel potential therapeutic target for breast cancer treatment.

KDM6B promotes activation of the oncogenic CDK4/6-pRB-E2F pathway by maintaining enhancer activity in MYCN-amplified neuroblastoma

The H3K27me2/me3 histone demethylase KDM6B is essential to neuroblastoma cell survival. However, the mechanism of KDM6B action remains poorly defined. We demonstrate that inhibition of KDM6B activity 1) reduces the chromatin accessibility of E2F target genes and MYCN, 2) selectively leads to an increase of H3K27me3 but a decrease of the enhancer mark H3K4me1 at the CTCF and BORIS binding sites, which may, consequently, disrupt the long-range chromatin interaction of MYCN and E2F target genes, and 3) phenocopies the transcriptome induced by the specific CDK4/6 inhibitor palbociclib. Overexpression of CDK4/6 or Rb1 knockout confers neuroblastoma cell resistance to both palbociclib and the KDM6 inhibitor GSK-J4. These data indicate that KDM6B promotes an oncogenic CDK4/6-pRB-E2F pathway in neuroblastoma cells via H3K27me3-dependent enhancer-promoter interactions, providing a rationale to target KDM6B for high-risk neuroblastoma.

Abstract LBA010: Therapeutic stimulation of nucleic acid receptor RIG-I enhances efficacy of kinase inhibitor treatment in oncogene-driven tumors

Abstract Introduction: A major challenge in the treatment of kinase driven tumors is the inevitable emergence of resistance and the limited efficacy of subsequent treatments including immunotherapy. Methods: To gain deeper insights into the adaptive processes of cells surviving kinase inhibition we studied the mechanisms enabling cell persistence despite targeted treatment across various in vitro and in vivo tumor models. This included transcriptomic profiles of EGFR-, BRAF-, HER2- or ALK-driven colon, melanoma and lung cancer cells and of melanoma patients during kinase inhibition. To investigate underlying mechanisms we employed CRISPR/Cas9 and overexpression experiments and stimulation of nucleic acid receptors (NARs) TLR3, cGAS and RIG-I with synthetic agonists to assess combination therapies. Finally, we used humanized tumor xenografts and a syngeneic EGFR-mutant lung cancer model to validate the inflammatory effects of kinase inhibition and the efficacy of adding RIG-I agonist IVT4 to kinase inhibition in vivo. Results: Analysis of dynamic transcriptomic changes during kinase inhibition by time-series RNA-seq of oncogene-dependent cells displayed rapid induction of an interferon gene signature and suppression of MYC- and E2F-target genes. This was accompanied by induction of p27 protein levels, cell cycle arrest and an increase of b-galactosidase positive cells, indicating a senescence-associated inflammatory response that occurred in a cell autonomous manner in vitro and in vivo. In vivo, EGFR inhibition also increased T-cells, but anti-PD-1 therapy following EGFR inhibitor pre-treatment did not prevent tumor recurrence. Interestingly, the kinase inhibitor induced interferon signature was independent of caspase activation and of the nucleic acid sensing machinery. Instead, functional experiments indicated that induction of interferon signaling was dependent on inhibition of MAPK signaling and subsequent IRF1 activation, providing a potential mechanism of action. Numerous innate immune pathway members including NARs MDA-5, TLR3 and RIG-I were upregulated, which we aimed to exploit therapeutically. Evaluation of NAR agonists showed that only stimulation of RIG-I with its agonist IVT4 evoked cytokine secretion and synergistically enhanced cell death in vitro. In humanized xenografts and syngenic immunocompetent mouse models adding IVT4 led to additional tumor shrinkage after EGFR-inhibition and decreased T cell exhaustion. Effects of IVT4 on tumor volumes were lower in immunodeficient xenografts and after NK- or CD8-cell depletion in the syngeneic model indicating that immunological influences in addition to tumor cell intrinsic effects contribute to the efficacy in vivo. Conclusion: Taken together, our findings show that kinase inhibition may promote tumor cell-autonomous, senescence-associated inflammatory signaling and may sensitize oncogene-driven tumors to therapeutic intervention using RIG-I agonist IVT4. Our results thus provide a novel treatment strategy and a basis to explore combining immunotherapy with kinase inhibition in the future. Citation Format: Johannes Brägelmann, Carina Lorenz, Sven Borchmann, Kazuya Nishii, Julia Wegner, Jenny Ostendorp, David Ast, Alena Heimsoeth, Philipp Lohneis, Thomas Zillinger, Roland Ullrich, Kadoaki Ohashi, Martin Schlee, Martin Sos. Therapeutic stimulation of nucleic acid receptor RIG-I enhances efficacy of kinase inhibitor treatment in oncogene-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA010.

CDC6: A novel canine tumour biomarker detected in circulating extracellular vesicles.

Circulating nucleic acids and extracellular vesicles (EV) represent novel biomarkers to diagnose cancer. The non‐invasive nature of these so‐called liquid biopsies provides an attractive alternative to tissue biopsy‐based cancer diagnostics. This study aimed to investigate if circulating cell cycle‐related E2F target transcripts can be used to diagnose tumours in canine tumour patients with different types of tumours. Furthermore, we assessed if these mRNAs are localised within circulating EV. We isolated total RNA from the plasma of 20 canine tumour patients and 20 healthy controls. Four E2F target genes (CDC6, DHFR, H2AFZ and ATAD2) were selected based on the analysis of published data of tumour samples available in public databases. We performed reverse transcription and quantitative real‐time PCR to analyse the plasma levels of selected E2F target transcripts. All four E2F target transcripts were detectable in the plasma of canine tumour patients. CDC6 mRNA levels were significantly higher in the plasma of canine tumour patients compared to healthy controls. A subset of canine tumour patient and healthy control plasma samples (n = 7) were subjected to size exclusion chromatography in order to validate association of the E2F target transcripts to circulating EV. For CDC6, EV analysis enhanced their detectability compared to total plasma analysis. In conclusion, our study reveals circulating CDC6 as a promising non‐invasive biomarker to diagnose canine tumours.

An EZH2 Gene Expression Signature Is Predictive of Differential Efficacy of Chemotherapy Irrespective of EZH2 Mutation Status in Patients with Follicular Lymphoma Treated within the Gallium Trial

An EZH2 Gene Expression Signature Is Predictive of Differential Efficacy of Chemotherapy Irrespective of EZH2 Mutation Status in Patients with Follicular Lymphoma Treated within the Gallium Trial

Distinct Pathogenesis of Clonal Hematopoiesis Revealed By Single Cell RNA Sequencing Integrated with Highly Sensitive Genotyping Method

BackgroundClonal hematopoiesis (CH) in apparently healthy individuals has been drawing an increasing attention of recent years due to its implication in the risk of hematopoietic malignancies and also in cardiovascular complications. However, our knowledge on CH has been largely based on genetic studies, while few functional analyses have been performed using human materials with most studies being confined to artificial models using mice. The major challenge here is the difficulty of isolating CH clones from wild-type (WT) cells in human bone marrow (BM) to elucidate the effect of CH-mutations.MethodsTo investigate cellular phenotypes of mutated and WT cells separately in CH, we developed a Fluidigm C1-based single cell-sequencing platform for simultaneous genotyping and gene expression analysis. We analyzed a total of 10,178 hematopoietic stem/progenitors (HSPCs) derived from BM of patients with (n=11) and without (n=17) CH.ResultsIn the analysis of HSPCs from CH(−) elderly individuals, we found a significant positive correlation between age and expression of gene sets implicated in inflammatory responses including TGFβ signalling and IL-6. We next analysed CH(+) samples, including those with mutations in TET2, DNMT3A, SF3B1 and IDH1 in which we investigated the pathways enriched in differentially expressed genes between mutated and unmutated cells. Regardless of mutation type, mutant cells showed an upregulation of genes implicated in an enhanced cell proliferation, while genes related to inflammatory responses were significantly downregulated. These results suggest that mutant HSPCs show an enhanced cell proliferation and an attenuated response to an inflammatory microenvironment in aged BM, compared with endogenous WT counterparts. Based on these observations, we further investigated the role of the BM microenvironment in CH, in which we compared the phenotype of WT cells in age-matched CH(+) and CH(−) cases. Compared with those from CH(−) cases, WT cells from these CH(+) cases showed an enhanced response to proinflammatory cytokines, including IL-6, interferons and TNF, suggesting a possibility that CH(+) BM might be characterized by a more enhanced inflammatory microenvironment, compared with CH(−) BM.To understand the different phenotype of WT cells between CH(+) and CH(−) cases, we next investigated a possible effect of mutant cells on endogenous WT cells. Such an effect was first documented in an IDH1-mutated CH case. IDH1-mutated cells showed a gene expression profile suggestive of an enhanced cell proliferation compared with endogenous IDH1-WT cells (WT IDH1). However, on the basis of comparison of gene expression between WT cells from CH(−) cases, it was better explained by suppressed proliferation of endogenous WT cells, rather than enhanced proliferation of mutant cells. In agreement with this, mouse BM cells treated with 2-hydroxyglutalate (2HG), an oncometabolite produced by IDH1-mutant cells, mimicked the endogenous WT cells in IDH1-mutated cases, including downregulated E2F target genes and upregulated inflammation-related genes, compared with control BM cells. A non-cell autonomous effect of mutations was also seen in cases with TET2-mutated CH, in which not only TET2-mutated cells but also endogenous WT cells exhibited a significantly different gene expression profile, compared with those from CH(−) cases. Of interest, when BM cells from WT Ly5.1/5.2 mice were co-transplanted with those from heterozygous conditional Tet2 knock-out (Ly5.2) or WT (Ly5.2) mice into lethally irradiated mice (Ly5.1) and flow-sorted Lin − WT donor competitor cells were analysed using single cell sequencing, the WT HSC-like cells co-transplanted with Tet2-mutant competitors exhibited enhanced cell proliferation and IFNα and IFNγ pathway genes, compared with those co-transplanted with WT competitors.ConclusionsTaken together, these results suggest that mutant cells in CH(+) BM have non-cell autonomous effects on endogenous WT cells, which might be responsible for an accelerated inflammatory microenvironment of aged BM, favor positive selection of CH-clones, and also affect the phenotype of endogenous WT cells, contributing to the pathogenesis of CH. DisclosuresNakagawa: Sumitomo Dainippon Pharma Oncology, Inc.: Research Funding. Inagaki: Sumitomo Dainippon Pharma Oncology, Inc.: Current Employment. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Yoda: Chordia Therapeutics Inc.: Research Funding. Ogawa: Kan Research Laboratory, Inc.: Consultancy, Research Funding; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding; ChordiaTherapeutics, Inc.: Consultancy, Research Funding; Ashahi Genomics: Current holder of individual stocks in a privately-held company; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding.

PRMT5 Inhibition Modulates E2F1 and P53 to Restore Cell Cycle Regulation and Drive DNA Damage Response in Ibrutinib-Resistant Mantle Cell Lymphoma

Introduction:Ibrutinib is widely used for relapsed/refractory mantle cell lymphoma (MCL), however nearly 1/3 of patients have primary resistance and the remaining patients will inevitably acquire resistance with poor overall survival. There is an urgent need for novel therapies targeting pro-survival signaling pathways triggered by ibrutinib resistance. Pharmacologic inhibition of Protein Arginine Methyltransferase 5 (PRMT5) represents a novel therapeutic approach to overcome ibrutinib resistant MCL (IR-MCL). PRMT5 has many essential functions in normal and malignant B cells, however in the context of IR-MCL the mechanisms of PRMT5 inhibitor mediated cell death have not been described. Genomic co-deletions of MTAP/CDKN2A are commonly detected in IR-MCL cells. MTAP deletion and subsequent accumulation of the metabolite MTA, sensitizes cells to further inhibition of PRMT5. Although the published literature defines this therapeutic rationale for targeting PRMT5 in cancers with co-deletion of MTAP/CDKN2A, the literature lacks studies specifically targeting PRMT5 in IR-MCL.Methods:Here we report the primary mechanism of PRMT5 inhibitor mediated cell death in the context of IR-MCL, utilizing a combination of Whole Exome Sequencing (WES), RNA-seq, protein expression, and cell cycle analysis (PI-DNA staining with flow cytometry) in 8 MCL cell lines and 3 patient derived xenografts (PDXs) of IR-MCL. Pharmacologic inhibition of PRMT5 was performed with two selective compounds: PRT-382 and C220 (Prelude Therapeutics). Inducible CRISPR/CAS9-gRNA was used to selectively knock out MTAP and CDKN2A.Results:Comparing MCL cell lines with primary ibrutinib resistance, we found an inverse correlation between PRT-382 ic50 and ibrutinib ic50 (Spearman: r = -0.94, p = 0.017). In MCL cell lines, low expression of MTAP also correlated with lower global Symmetric Di-Methyl Arginine (SDMA) and enhanced sensitivity to C220 respectively (Pearson: r = 0.84, p = 0.01; r = 0.87, p = 0.01). Genomic deletion of MTAP in a cell line with primary ibrutinib resistance (Jeko MTAP-WT) enhanced sensitivity to PRMT5i, whereas MTAP knock-in (KI) in CCMCL, an MTAP-null cell line, decreased sensitivity to PRMT5i. The treatment of Jeko-MTAP-KI and CCMCL-MTAP-KO with MTA, further confirmed that genomic deletion of MTAP confers enhanced vulnerability to PRMT5i in MCL cell lines.In a PDX generated in our lab from an MCL patient with acquired IR, treatment with PRT-382 decreased tumor infiltration of multiple organs and ki-67 compared to ibrutinib or control (H&E and IHC staining). To mechanistically address the nature of the anti-tumor activity of PRMT5i in IR-MCL, we treated PDX mice with PRT-382 or vehicle control for 2 weeks to obtain ex-vivo samples for NGS methods. Splenic MCL tumor cells (Hu-CD19+) where subjected to transcriptomic profiling by RNA-seq (n=3 per Tx group). RNA-seq revealed 1014 down and 1124 up differentially expressed genes (DEGs) (q < 0.01, |log2FC| > 0.05). Gene Set Enrichment Analysis (GSEA) identified activation of the p53 pathway (NES = 1.73), with repression of E2F targets, G2M checkpoints, Epithelial to Mesenchymal Transition (EMT), and PRMT5 targets among the top gene sets modulated with PRMT5i (NES = -2.51, -2.26, -2.06, -1.79). Comparing genomic alterations across MCL cell lines and 3 PDXs of IR-MCL, co-deletion of MTAP/CDKN2A and/or a functional/WT copy of TP53 defines a therapeutic vulnerability to PRMT5i in 9 out of 11 cases (82%). In MCL, PRMT5i resulted in p53-dependent G1/S cell cycle arrest and p53-dependent DNA damage induced apoptosis by transcriptional activation of the p53 target genes (CDKN1A, BBC3, BAX, PHLDA3, andSESN1). Western blot analysis confirmed E2F target gene repression (TK1, CCNA2, CHK1, CDK1) and accumulation of DNA damage response proteins (cleaved PARP, cleaved CASP3, p-H2AX) that are further enhanced with combination of doxorubicin.Conclusion:Pharmacologic inhibition of PRMT5 with the clinical-grade, novel small molecule inhibitor (PRT543, Prelude Therapeutics) merits further investigation in ongoing clinical trials (NCT03886831, clinicaltrials.gov), specifically for the treatment of IR-MCL with deletion of MTAP and prior to the genomic mutation of TP53. We are currently exploring novel combinations to maximize the therapeutic potential of PRMT5 inhibition in this disease. DisclosuresByrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees. Vaddi: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Scherle: Prelude Therapeutics: Current Employment, Current equity holder in publicly-traded company. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy.

Drivers of transcriptional variance in human intestinal epithelial organoids.

Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.

Transcription Factor Homeobox D9 Drives the Malignant Phenotype of HPV18-Positive Cervical Cancer Cells via Binding to the Viral Early Promoter.

Simple SummaryTranscription factor homeobox D9 (HOXD9) was previously reported to bind to the P97 promoter of HPV16 to induce viral E6/E7 oncogenes. In this article, we investigated whether HOXD9 regulated the P105 promoter of HPV18 and examined the role of HOXD9 in intracellular signaling of cervical cancer (CC). HOXD9 was directly bound to the P105 promoter and regulated the expression of E6/E7 genes of HPV18. The HOXD9 knockdown suppressed the E6/E7 gene expression in HPV18-positive cervical cancer cells. It decreased the expression of E6, activated the p53 pathway, and induced apoptosis. In addition, downregulation of the E7 gene expression activated the Rb pathway, causing G1 arrest in the cell cycle and markedly suppressing cell proliferation. Our results indicate that HOXD9 has pivotal roles in the proliferation and immortalization of HPV18-positive cervical cancer cells through activating the P105 promoter.Persistent infections with two types of human papillomaviruses (HPV), HPV16 and HPV18, are the most common cause of cervical cancer (CC). Two viral early genes, E6 and E7, are associated with tumor development, and expressions of E6 and E7 are primarily regulated by a single viral promoter: P97 in HPV16 and P105 in HPV18. We previously demonstrated that the homeobox D9 (HOXD9) transcription factor is responsible for the malignancy of HPV16-positive CC cell lines via binding to the P97 promoter. Here, we investigated whether HOXD9 is also involved in the regulation of the P105 promoter using two HPV18-positive CC cell lines, SKG-I and HeLa. Following the HOXD9 knockdown, cell viability was significantly reduced, and E6 expression was suppressed and was accompanied by increased protein levels of P53, while mRNA levels of TP53 did not change. E7 expression was also downregulated and, while mRNA levels of RB1 and E2F were unchanged, mRNA levels of E2F-target genes, MCM2 and PCNA, were decreased, which indicates that the HOXD9 knockdown downregulates E7 expression, thus leading to an inactivation of E2F and the cell-cycle arrest. Chromatin immunoprecipitation and promoter reporter assays confirmed that HOXD9 is directly associated with the P105 promoter. Collectively, our results reveal that HOXD9 drives the HPV18 early promoter activity to promote proliferation and immortalization of the CC cells.

H2AFZ: A Novel Prognostic Marker in Canine Melanoma and a Predictive Marker for Resistance to CDK4/6 Inhibitor Treatment.

Uncontrolled proliferation is a key feature of tumor progression and malignancy. This suggests that cell-cycle related factors could be exploited as cancer biomarkers and that pathways specifically involved in the cell cycle, such as the Rb-E2F pathway, could be targeted as an effective anti-tumor therapy. We investigated 34 formalin-fixed paraffin-embedded (FFPE) tissue samples of canine cutaneous melanocytoma, cutaneous melanoma, and oral melanoma. Corresponding clinical follow-up data were used to determine the prognostic value of the mRNA expression levels of several cell cycle regulated E2F target genes (E2F1, DHFR, CDC6, ATAD2, MCM2, H2AFZ, GINS2, and survivin/BIRC5). Moreover, using four canine melanoma cell lines, we explored the possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor (Palbociclib) as a potential anti-cancer therapy. We investigated the expression levels of the same E2F target gene transcripts before and after treatment to determine the potential utility of these molecules as predictive markers. The E2F target gene H2AFZ was expressed in 91.43% of the primary tumors and H2AFZ expression was significantly higher in cases with unfavorable clinical outcome. Among the other tested genes, survivin/BIRC5 showed as well-promising results as a prognostic marker in canine melanoma. Three of the four tested melanoma cell lines were sensitive to the CDK4/6 inhibitor. The resistant cell line displayed higher expression levels of H2AFZ before treatment compared to the CDK4/6 inhibitor-sensitive cell lines. The present results suggest that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma and that H2AFZ could serve as a prognostic and predictive marker for patient selection.

The p53 transcriptional response across tumor types reveals core and senescence-specific signatures modulated by long noncoding RNAs

The p53 pathway is a universal tumor suppressor mechanism that limits tumor progression by triggering apoptosis or permanent cell cycle arrest, called senescence. In recent years, efforts to reactivate p53 function in cancer have proven to be a successful therapeutic strategy in murine models and have gained traction with the development of a range of small molecules targeting mutant p53. However, knowledge of the downstream mediators of p53 reactivation in different oncogenic contexts has been limited. Here, we utilized a panel of murine cancer cell lines from three distinct tumor types susceptible to alternative outcomes following p53 restoration to define unique and shared p53 transcriptional signatures. While we found that the majority of p53-bound sites and p53-responsive transcripts are tumor-type specific, analysis of shared targets identified a core signature of genes activated by p53 across all contexts. Furthermore, we identified repression of E2F and Myc target genes as a key feature of senescence. Characterization of p53-induced transcripts revealed core and senescence-specific long noncoding RNAs (lncRNAs) that are predominantly chromatin associated and whose production is coupled to cis-regulatory activities. Functional investigation of the contributions of p53-induced lncRNAs to p53-dependent outcomes highlighted Pvt1b, the p53-dependent isoform of Pvt1, as a mediator of p53-dependent senescence via Myc repression. Inhibition of Pvt1b led to decreased activation of senescence markers and increased levels of markers of proliferation. These findings shed light on the core and outcome-specific p53 restoration signatures across different oncogenic contexts and underscore the key role of the p53-Pvt1b-Myc regulatory axis in mediating proliferative arrest.

QSOX2 Is an E2F1 Target Gene and a Novel Serum Biomarker for Monitoring Tumor Growth and Predicting Survival in Advanced NSCLC.

BackgroundQuiescin Q6 sulfhydryl oxidase 2 (QSOX2), an enzyme that can be directly secreted into the extracellular space, is known to be associated with oxidative protein folding. However, whether QSOX2 is abnormally expressed in non-small cell lung cancer (NSCLC) and its role in tumor growth remains unclear.MethodsReal-time quantitative PCR (qPCR), immunohistochemistry (IHC), bioinformatics analyses were applied to analyze the expression pattern and prognostic significance of QSOX2 in NSCLC. Xenografts model, enzyme-linked immunosorbent assays (ELISA), western blot analysis (WB), and IHC were preformed to examine in vivo tumor suppression and intracellular and extracellular expression of QSOX2. Flow cytometry, WB and qPCR analyses were used to elucidate the role of QSOX2 in cell cycle regulation. Chromatin immunoprecipitation assay (ChIP) assay and Dual-Luciferase reporter assay were employed to investigate transcriptional regulation of QSOX2 by E2F Transcription Factor 1 (E2F1).ResultsQuiescin sulfhydryl oxidase 2 was significantly overexpressed in NSCLC and associated with poor survival in advanced-stage patients. The intracellular and extracellular expression of QSOX2 by tumor cells markedly decreased after anti-cancer therapy in vitro, in vivo and in the clinic. Moreover, QSOX2 silencing in NSCLC cell lines resulted in inhibition of cancer cell proliferation, induction of apoptosis, and decreased expression of cell division-related genes (CENPF and NUSAP1) and Wnt pathway activators (PRRX2 and Nuc-β-catenin). Mechanistically, QSOX2 was expressed periodically during cell cycle and directly regulated by E2F1.ConclusionsOur findings demonstrate that QSOX2 is directly regulated by E2F1 in the cell cycle, which is essential for the proliferation of NSCLC cells. Furthermore, QSOX2 is a prognostic indicator for NSCLC and may be developed into a biomarker for monitoring tumor burden and therapeutic progress.

Loss of MGA repression mediated by an atypical polycomb complex promotes tumor progression and invasiveness

MGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated Mga in two mouse models of non-small cell lung cancer using a CRISPR-based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of non-canonical Polycomb ncPRC1.6 targets, including genes involved in metastasis and meiosis. Moreover, MGA deletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA-MAX, E2F6, and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes these ncPRC1.6 subunits. Lastly, we report that MGA loss also induces a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F target genes mediated by MGA-MAX associated with a non-canonical Polycomb complex.

AP-2α-Mediated Activation of E2F and EZH2 Drives Melanoma Metastasis.

In melanoma metastasis, the role of the AP-2α transcription factor, which is encoded by TFAP2A, is controversial as some findings have suggested tumor suppressor activity while other studies have shown high TFAP2A expression in node-positive melanoma associated with poor prognosis. Here we demonstrate that AP-2α facilitates melanoma metastasis through transcriptional activation of genes within the E2F pathway including EZH2. A BioID screen found that AP-2α interacts with members of the nucleosome remodeling and deacetylase (NuRD) complex. Loss of AP-2α removed activating chromatin marks in the promoters of EZH2 and other E2F target genes through activation of the NuRD repression complex. In melanoma cells, treatment with tazemetostat, an FDA-approved and highly specific EZH2 inhibitor, substantially reduced anchorage-independent colony formation and demonstrated heritable antimetastatic effects, which were dependent on AP-2α. Single-cell RNA sequencing analysis of a metastatic melanoma mouse model revealed hyperexpansion of Tfap2a High/E2F-activated cell populations in transformed melanoma relative to progenitor melanocyte stem cells. These findings demonstrate that melanoma metastasis is driven by the AP-2α/EZH2 pathway and suggest that AP-2α expression can be used as a biomarker to predict responsiveness to EZH2 inhibitors for the treatment of advanced melanomas. SIGNIFICANCE: AP-2α drives melanoma metastasis by upregulating E2F pathway genes including EZH2 through inhibition of the NuRD repression complex, serving as a biomarker to predict responsiveness to EZH2 inhibitors.

CARM1/PRMT4 As a Novel Therapeutic Target for AML

Chromatin modifying enzymes, and specifically the protein arginine methyltransferases (PRMTs) have emerged as important targets in cancer. PRMT4, also known as CARM1, is overexpressed in a number of cancers, including breast, prostate, pancreatic, and lung cancer. We have reported the overexpression of PRMT4 in the context of acute myeloid leukemia (AML), showing that more than 70% of cytogenetically normal AML patients have up-regulation of PRMT4. Here, we investigated the role of PRMT4 in normal hematopoiesis and leukemia development.In order to study the role of PRMT4 in normal hematopoiesis, Prmt4 -floxed mice were crossed with Vav1-cre mice purchased from the Jackson Laboratory. Inducible Prmt4 conditional KO mice were generated by crossing Prmt4 -floxed mice with Mx1-Cre mice and Prmt4 gene excision was induced by poly(I:C), given by three i.p. injections at a dose of 10 mg/kg. Using this hematopoietic specific knockout system, we show that loss of PRMT4 has little effect on normal hematopoiesis, but promotes the differentiation of hematopoietic stem and progenitor cells. Next we evaluated the role of PRMT4 in leukemia initiation and maintenance using leukemia transplantation models driven by fusion oncoproteins. Strikingly, the knockout of PRMT4 completely abrogates leukemia initiation and maintenance in fetal liver transplantation models driven by the AML1-ETO or MLL-AF9 fusion proteins.We further characterized the mechanism for the leukemia-specific dependence on PRMT4 using leukemia cell lines lentiviral vectors that express shRNAs directed against PRMT4. We knocked down PRMT4 in four leukemia cell lines or normal human cord-blood derived CD34+ cells and found that knockdown of PRMT4 impairs cell cycle progression, induces apoptosis, and down-regulates E2F target genes, identifying several mechanisms for the leukemia-specific dependence on PRMT4. Gene set enrichment analysis showed that all four leukemia cell lines with knockdown of PRMT4 showed a significant down-regulation of MYC and E2F target genes compared to the scrambled control. Differentially regulated pathways included Cell cycle, DNA replication, and Homologous recombination. Finally, we utilized inhibitors of the Type I arginine methyltransferase family in order to validate the selective inhibition of PRMT4 in leukemia cells and patient cell cells.The PRMT4 conditional knockout mice, PRMT4 knockdown in leukemia cell lines, and chemical inhibition of PRMT4, all suggest that loss of PRMT4 protein levels or activity has a selective effect on leukemia cells compared to normal hematopoietic stem and progenitor cells. Collectively this work suggests that targeting PRMT4 activity may be an effective therapeutic strategy for AML, with minimal effects on normal hematopoiesis. DisclosuresWatts:Jazz Pharmaceuticals: Consultancy, Speakers Bureau.

Treatment with the Novel TOPK Inhibitor OTS514 Exhibits Potent Anti-Myeloma Activity in Pre-Clinical Models

Multiple myeloma (MM) continues to be considered incurable, necessitating new drug discovery. TOPK, a mitotic kinase, is upregulated in many human cancers, including MM, and is associated with proliferation of tumor cells, maintenance of cancer stem cells, and poor patient prognosis. Gene expression profiling shows elevated TOPK transcript levels in the plasma cells (PC) of patients with MM relative to healthy individuals, but not in PC derived from patients diagnosed with MM precursor states MGUS or smoldering myeloma. Western blot analysis of cells from bone marrow aspirates (BMAs) from patients with frank MM shows strong TOPK protein levels in the enriched CD138+ population (MM PCs), but not in the CD138- fraction or in peripheral blood mononuclear cells (PBMCs). In addition, TOPK protein is robustly expressed among all human myeloma cell lines (HMCLs) tested. In this study, we targeted this candidate oncogene with the selective inhibitor OTS514 to assess the contribution of TOPK to MM cell survival.Treatment of HMCLs with the TOPK inhibitor OTS514 causes rapid cleavage of caspase 3 and PARP, and subsequent DNA fragmentation, indicative of a robust apoptotic response. Viability assays reveal potent cell death in a panel of 9 HMCLs, with 72-hour IC50s ranging from 11-29 nM. Importantly, no apparent resistance to TOPK inhibition was observed among six p53-mutant cell lines tested. OTS514 treatment of total marrow mononuclear cells derived from BMAs from patients with MM resulted in robust cell death of CD138+ PC, as measured by flow cytometric analysis of Annexin-V/propidium iodide-stained cells, whereas CD138- cell populations were largely protected from apoptotic death, corroborating the relative lack of TOPK expression in non-PC and suggesting a highly favorable therapeutic index. OTS514 treatment also prevented CD138+ cell outgrowth from MM patients' PBMCs stimulated with IL-3 and IL-6, suggesting TOPK may play a role in maintenance of the putative myeloma stem cell populations.TOPK inhibition causes time- and dose-dependent decreases in both TOPK auto-phosphorylation and total protein levels. Protein expression of known TOPK target FOXM1 is also lost, as well as expression of pro-survival factors, including phospho-Akt. Microarray analysis of H929 cells revealed elevated expression of 73 small nucleolar RNAs (snoRNA), which are involved in ribosome maturation, and this is likely the result of a nucleolar/ribosomal stress response caused by cell cycle perturbation. Gene Set Enrichment Analysis revealed reduced expression of E2F1 target genes and western blot analysis showed a time-dependent decrease in levels of E2F1 in response to OTS514 treatment, suggesting a role of TOPK at the G1 checkpoint.The anti-myeloma activity of OTS514 was also explored in combination with the immunomodulatory agent lenalidomide and found to be exquisitely synergistic; Chou-Talalay combination indices at IC50, IC75, and IC90 were <1 among all HMCL tested. Combination treatment with OTS514 and lenalidomide also led to additive loss of the key MM cell survival proteins IKAROS and IRF4.The modified TOPK inhibitor OTS964, which is more amenable to oral administration, was evaluated in an animal model. H929 cells were xenografted subcutaneously into NOD/SCID mice and daily drug gavage was given at 50 or 100 mg/kg for 14 days after tumors reached 100 mm3. After 14 days of treatment, average tumor volume was reduced by 81% in mice treated with the 100 mg/kg dose of OTS964 relative to controls. The drug was well-tolerated, with minimal and reversible body weight loss in the group receiving the highest dose.TOPK is emerging as a key oncogene in several cancer types. Our data strongly support further study of the mechanistic contribution of TOPK to the development of MM and its inhibition as a potential therapy for MM. DisclosuresPark:OncoTherapy Science, Inc.: Consultancy. Jakubowiak:Amgen Inc., BMS, Celgene, Janssen, Karypharm, Millennium-Takeda, Sanofi, SkylineDX: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; University of Chicago: Employment.

PARP and CDK4/6 Inhibitor Combination Therapy Induces Apoptosis and Suppresses Neuroendocrine Differentiation in Prostate Cancer.

We analyzed the efficacy and mechanistic interactions of PARP inhibition (PARPi; olaparib) and CDK4/6 inhibition (CDK4/6i; palbociclib or abemaciclib) combination therapy in castration-resistant prostate cancer (CRPC) and neuroendocrine prostate cancer (NEPC) models. We demonstrated that combined olaparib and palbociblib or abemaciclib treatment resulted in synergistic suppression of the p-Rb1-E2F1 signaling axis at the transcriptional and posttranslational levels, leading to disruption of cell-cycle progression and inhibition of E2F1 gene targets, including genes involved in DDR signaling/damage repair, antiapoptotic BCL-2 family members (BCL-2 and MCL-1), CDK1, and neuroendocrine differentiation (NED) markers in vitro and in vivo In addition, olaparib + palbociclib or olaparib + abemaciclib combination treatment resulted in significantly greater growth inhibition and apoptosis than either single agent alone. We further showed that PARPi and CDK4/6i combination treatment-induced CDK1 inhibition suppressed p-S70-BCL-2 and increased caspase cleavage, while CDK1 overexpression effectively prevented the downregulation of p-S70-BCL-2 and largely rescued the combination treatment-induced cytotoxicity. Our study defines a novel combination treatment strategy for CRPC and NEPC and demonstrates that combination PARPi and CDK4/6i synergistically promotes suppression of the p-Rb1-E2F1 axis and E2F1 target genes, including CDK1 and NED proteins, leading to growth inhibition and increased apoptosis in vitro and in vivo Taken together, our results provide a molecular rationale for PARPi and CDK4/6i combination therapy and reveal mechanism-based clinical trial opportunities for men with NEPC.

A Novel Three-Gene Score as a Predictive Biomarker for Pathologically Complete Response after Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

Simple SummaryNeoadjuvant chemotherapy is now a standard of care not only to decrease tumor size for breast conserving operation but also to assess drug response of an in situ cancer. Although the triple-negative subtype typically responds better compared to the other subtypes, a pathological complete response, which is a surrogate of survival, is achieved in less than half of the cases. For the most efficient patient selection, and avoiding unnecessary side effects and financial toxicity, an accurate predictive biomarker is urgently needed. We developed a novel three-gene score that associated with immune cell infiltration and pathological complete response not only in the training cohort but also in the validation triple-negative cohort. High-score TNBC was significantly associated with better survival in patients who received chemotherapy but not in patients who did not receive chemotherapy. Our score is a predictive and prognostic biomarker of response to neoadjuvant chemotherapy in triple-negative breast cancer patients.Although triple-negative breast cancer (TNBC) typically responds better to neoadjuvant chemotherapy (NAC) compared to the other subtypes, a pathological complete response (pCR) is achieved in less than half of the cases. We established a novel three-gene score using genes based on the E2F target gene set that identified pCR after NAC, which showed robust performance in both training and validation cohorts (total of n = 3862 breast cancer patients). We found that the three-gene score was elevated in TNBC compared to the other subtypes. A high score was associated with Nottingham histological grade 3 in TNBC. Across multiple cohorts, high-score TNBC enriched not only E2F targets but also G2M checkpoint and mitotic spindle, which are all cell proliferation-related gene sets. High-score TNBC was associated with homologous recombination deficiency, high mutation load, and high infiltration of Th1, Th2, and gamma-delta T cells. However, the score did not correlate with drug sensitivity for paclitaxel, 5-fluorouracil, cyclophosphamide, and doxorubicin in TNBC human cell lines. High-score TNBC was significantly associated with a high rate of pCR not only in the training cohort but also in the validation cohorts. High-score TNBC was significantly associated with better survival in patients who received chemotherapy but not in patients who did not receive chemotherapy. The three-gene score is associated with a high mutation rate, immune cell infiltration, and predicts response to NAC in TNBC.