E2F Transcriptional Activity Research Articles

AB067. New drug development for the use of PARP1-E2F1 transcriptional inhibitors in the treatment of glioblastoma.

Glioblastoma (GBM) is the most malignant brain tumor and ranks among the most lethal of all human cancers, without improvements in survival over the last 30 years. Data obtained in our group suggest that PARP1, a well-known DNA-repairing protein, could also play a key role in the regulation of cell cycle through its interaction with the transcription factor E2F1. Therefore, considering that most oncogenic processes are associated with cell cycle deregulation, we hypothesized that disruption of PARP1-E2F1 interaction would provide a novel therapeutic approach to different types of cancer. The identification of novel compounds disrupting PARP1-E2F1 interaction was carried out by combining in silico and in vitro screening, using a rational drug design. The virtual screen was performed using a molecular library of several million compounds at the selected target site, using AtomNet® (Atomwise, San Francisco, CA, USA), the first deep learning neural network for structure-based drug design and discovery. Since there is no complete structural information of the PARP1-E2F1 protein-protein interaction, a homologous structure of the BRCT domain of BRCA1 complex with the phospho-peptide (PDBID: 1T2V) was used to identify the potential binding interface of BRCT domain of PARP-1 (PDBID: 2COK) and the E2F1 protein. Top scoring compounds were clustered and filtered to obtain a final subset of 83 compounds that were incorporated to our in vitro screening, which included both transcriptional E2F1 activity and survival studies. Complete culture medium supplemented with the compounds selected in the in silico screening (10 μM) were added and incubated for 24 hours. E2F1 activity was observed by measuring luminescence. For the viability assay, the fluorescence reading was performed (excitation 544 nm and emission 590 nm). The in silico and in vitro screening resulted in 12 compounds that inhibited E2F1 transcriptional activity and significantly reduced cell number. The highest inhibition of both E2F1 transcriptional activity and cell growth was observed with compound 3797, which was selected for further studies. Both in silico and in vitro results indicate that inhibition of PARP1-E2F1 transcriptional activity may provide a new rationale for designing novel therapeutic approaches for the treatment of GBM.

Abstract 5785: Shedding light on PARP1-E2F1 interaction by Cryo-EM

Abstract Cell division is orchestrated by a complex network of interactions between proteins involved in numerous signaling pathways that control cell proliferation. One of these main players is the E2F family of transcription factors. The role of these key players is to regulate the expression of several genes important in cell proliferation, particularly those involved in progression through G1 and into the S-phase of the cell cycle. In fact, cell cycle deregulation is a hallmark of cancer, and multiple studies have highlighted the critical role of E2F hyperactivation in tumor progression, angiogenesis, and metastasis. However, targeting a master regulator of cell growth will also have a pernicious effect on non-cancerous cells, thus limiting its applications. In order to overwhelm these restrictions, we propose a therapeutic approach in which E2F transcriptional activity is specifically reduced in cells with hyperactivation of the pathway, such as cancer cells.We and others have shown that poly (ADP-ribose) polymerase 1 (PARP1) is a transcriptional co-activator of E2F1 and, owing to this, loss of PARP results in cell cycle re-regulation and reduction of tumor growth. Interestingly, this effect of PARP1 does not depend on its enzymatic activity, thus opening a completely new strategic approach. Furthermore, considering that most oncogenic processes are associated with cell cycle deregulation, disruption of PARP1-E2F1 interaction could provide a new therapeutic target with a wide spectrum of indications in cancer. Currently, we lack comprehensive structural details regarding the PARP-1/E2F1 protein-protein interaction. To address this, we used cryo-EM to investigate both the standalone structure of PARP1 (full length) and its interaction with E2F1. This approach yielded high-resolution structural insights into the PARP1-E2F1 complex, enabling precise identification of the protein-protein interaction interface (PPI). Through this exploration of structural and molecular determinants governing this interaction, we aim to develop innovative strategies for designing drugs targeted at disrupting this complex. This work was supported by Agencia Estatal de Investigación (AEl/10.13039/501100011033), Xunta de Galicia (GPC GI-1862, ED431B 2020/26; ED431G 2019/02) and European Regional Development Fund-ERDF. Citation Format: Pablo Iglesias, Ester Casajus-Pelegay, Maria Moreno-Morcillo, Lara González-Rendo, Laura Porres-Ventin, Victor M. Arce, Rafael Fernandez-Leiro, Jose A. Costoya. Shedding light on PARP1-E2F1 interaction by Cryo-EM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5785.

Regulation of Cell Cycle Progression through RB Phosphorylation by Nilotinib and AT-9283 in Human Melanoma A375P Cells.

BCR-ABL tyrosine kinase inhibitors are commonly employed for the treatment of chronic myeloid leukemia, yet their impact on human malignant melanoma remains uncertain. In this study, we delved into the underlying mechanisms of specific BCR-ABL tyrosine kinase inhibitors (imatinib, nilotinib, ZM-306416, and AT-9283) in human melanoma A375P cells. We first evaluated the influence of these inhibitors on cell growth using cell proliferation and wound-healing assays. Subsequently, we scrutinized cell cycle regulation in drug-treated A375P cells using flow cytometry and Western blot assays. Notably, imatinib, nilotinib, ZM-306416, and AT-9283 significantly reduced cell proliferation and migration in A375P cells. In particular, nilotinib and AT-9283 impeded the G1/S transition of the cell cycle by down-regulating cell cycle-associated proteins, including cyclin E, cyclin A, and CDK2. Moreover, these inhibitors reduced RB phosphorylation, subsequently inhibiting E2F transcriptional activity. Consequently, the expression of the E2F target genes (CCNA2, CCNE1, POLA1, and TK-1) was markedly suppressed in nilotinib and AT9283-treated A375P cells. In summary, our findings suggest that BCR-ABL tyrosine kinase inhibitors may regulate the G1-to-S transition in human melanoma A375P cells by modulating the RB-E2F complex.

Abstract C062: Defining the interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in American men. Non-Hispanic Black men are 1.8 times more likely to develop PCa and 2.3 times more likely to die from PCa. Tumor suppressor retinoblastoma (RB1) is frequently deleted in PCa. RB represses the transcriptional activity of E2Fs and the phosphorylation of RB by CDK4/6 results in dissociation of the complex and a subsequent increase in E2F transcriptional activity, leading to cell cycle progression. RB loss is associated with tumor aggressiveness occurring in 10-15% of castration-resistant prostate cancer (CRPC) tumors and corresponds to a decrease in overall survival. RB depletion leads to increased expression of DNA repair proteins due to elevated transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in DNA repair. Elevated PARP-1 enzymatic activity is associated with PCa progression and correlates with poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, however the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry. Defining the interplay between PARP and RB/E2F is likely to contribute to the identification of novel treatment strategies and/or biomarker response. Canonically, E2F1-3 are transcription factors that positively regulate gene expression programs associated with pro-proliferative signals. Prior research by other groups has shown that E2F target genes expression is elevated in tumors excised from Black men compared to Asian and White men. There was an increase in germline RAD51 mutations observed. RAD51 is an E2F target gene involved in DDR, therefore attenuating PARP enzymatic activity may be a potential mode of targeting this axis. These data suggest that there may be differential DNA damage response (DDR) in African American men driven by aberrations in the RB/E2F signaling axis, however the mechanism is yet to be fully explored. Analysis of publicly available patient datasets were performed and demonstrate that RB1 deletions is more frequent than RB1/BRCA2 co-deletion in metastatic PCa. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 mRNA and PARP-1 protein are elevated upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. In hormone-depleted conditions, results suggest a differential response between RB depleted models compared to the control when challenged with PARP inhibition. Furthermore, an IC50 dose of Olaparib elicits differential PARP enzymatic response in the RB1 depleted model. RB1 loss is associated with an enrichment in E2F1 binding at the PARP-1 locus. Future studies have been designed to determine the impact of the RB/E2F pathway alterations on downstream consequences PARP enzymatic function and establish the contribution of PARP and the RB/E2F axis on racial disparity in PCa. Citation Format: Latese Evans. Defining the interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C062.

Sequential activation of E2F via Rb degradation and c-Myc drives resistance to CDK4/6 inhibitors in breast cancer

Cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i) are key therapeutic agents in the management of metastatic hormone-receptor-positive breast cancer. However, the emergence of drug resistance limits their long-term efficacy. Here, we show that breast cancer cells develop CDK4/6i resistance via a sequential two-step process of E2F activation. This process entails retinoblastoma (Rb)-protein degradation, followed by c-Myc-mediated amplification of E2F transcriptional activity. CDK4/6i treatment halts cell proliferation in an Rb-dependent manner but dramatically reduces Rb-protein levels. However, this reduction in Rb levels insufficiently induces E2F activity. To develop CDK4/6i resistance, upregulation or activating mutations in mitogenic or hormone signaling are required to stabilize c-Myc levels, thereby augmenting E2F activity. Our analysis of pre-treatment tumor samples reveals a strong correlation between c-Myc levels, rather than Rb levels, and poor therapeutic outcomes after CDK4/6i treatment. Moreover, we propose that proteasome inhibitors can potentially reverse CDK4/6i resistance by restoring Rb levels.

Abstract B063: Interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Retinoblastoma (RB) protein is a tumor suppressor that represses the transcriptional activity of E2Fs by forming an RB-E2F repressor complex. The phosphorylation of RB by CDK4/6 results in dissociation of the complex and subsequent E2F transcriptional activity, leading to cell cycle progression. RB loss occurs in 10-15% of castration-resistant prostate cancer (CRPC) which is associated tumor aggressiveness and poor clinical outcomes. RB loss is also associated differential treatment response to an array of treatment modalities, including hormone therapy and DNA damage inducing chemotherapy. RB depletion leads to enhanced expression of DNA repair proteins due to increased transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in multiple nuclear processes including DNA repair, transcriptional regulation, and chromatin dynamics. Increased PARP-1 activity is associated with and correlates to poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, but the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry, which may yield novel treatment strategies and/or biomarkers of response. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 protein increases upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. Results suggest a differential response between control and RB1 depleted cells when treated with Olaparib. RB1depleted cells also exhibit differential PARP activity in response to treatment with the IC50 dose of Olaparib. Future studies have been designed to evaluate the impact of RB alteration and manipulation of PARP activity on biological and molecular processes that are governed by RB/E2F and PARP. Citation Format: Latese Evans, Moriah Cunningham, Jasibel Vasquez Gonzalez, Matthew Schiewer. Interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B063.

The transcription factor E2F1 controls the GLP-1 receptor pathway in pancreatic β cells.

The glucagon-like peptide 1 (Glp-1) has emerged as a hormone with broad pharmacological potential in type 2 diabetes (T2D) treatment, notably by improving β cell functions. The cell-cycle regulator and transcription factor E2f1 is involved in glucose homeostasis by modulating β cell mass and function. Here, we report that β cell-specific genetic ablation of E2f1 (E2f1β-/-) impairs glucose homeostasis associated with decreased expression of the Glp-1 receptor (Glp1r) in E2f1β-/- pancreatic islets. Pharmacological inhibition of E2F1 transcriptional activity in nondiabetic human islets decreases GLP1R levels and blunts the incretin effect of GLP1R agonist exendin-4 (ex-4) on insulin secretion. Overexpressing E2f1 in pancreatic β cells increases Glp1r expression associated with enhanced insulin secretion mediated by ex-4. Interestingly, ex-4 induces retinoblastoma protein (pRb) phosphorylation and E2f1 transcriptional activity. Our findings reveal critical roles for E2f1 in β cell function and suggest molecular crosstalk between the E2F1/pRb and GLP1R signaling pathways.

Deltex E3 ubiquitin ligase 3 inhibits colorectal cancer cell growth and regulates cell cycle progression via upregulating E2F transcription factor 1

BackgroundThe mortality rate of colorectal cancer (CRC) remains high in developing countries. Interventions that can inhibit the proliferation of tumor cells represent promising strategies in CRC treatment. Deltex E3 ubiquitin ligase 3 (DTX3) plays an essential role in tumor development and may predict the outcome of cancer patients. This study aimed to investigate the regulatory mechanisms of DTX3 in CRC progression.Methods and resultsThe expression of DTX3 was significantly downregulated in CRC tissues relative to normal colorectal tissues. DTX3 overexpression inhibited, while DTX3 knockout promoted the colony-forming capacity and proliferation of CRC cells. E2F transcription factor 1 (E2F1) is a key mediator of cell cycle progression that participates in the progression, metastasis, and chemoresistance of CRC. Further analysis revealed that DTX3 regulated the transcriptional activity of E2F1 in CRC cells. The transcription by E2F1 was significantly reduced with the increase in the cellular level of DTX3, while DTX3 knockout exerted an opposite effect. DTX3 knockout also increased the expression of E2F1 target genes involved in cell cycle progression, CDC2 and Cyclin D3, while PD 0332991, an inhibitor of E2F1 transcription, inhibited the expression of both proteins.ConclusionsIn conclusion, DTX3 regulated CRC cell growth via regulating E2F1 and its downstream genes. These findings support further exploration of DTX3 as a potential therapeutic target for CRC.

Highly selective and potent p16-CDK-RB-E2F targeted oncolytic virus therapies.

e14543 Background: Growth factor and p16-CDK-RB-E2F pathway components are mutated in almost all cancers, resulting in oncogenic E2F transcriptional activity that drives uncontrolled proliferation. Chemotherapies indiscriminately kill all proliferating cells, resulting in limiting toxicities. CDK inhibitors are more targeted but cytostatic as opposed to tumoricidal. E2F was discovered as a factor that transcribes adenovirus (Ad) E2 and cellular genes to drive viral replication. Ad E1A binds to RB, which activates E2F. On this basis, viruses with E1A-RB binding mutations are being evaluated in the clinic as oncolytic viruses (OVs) that selectively replicate in and kill RB mutant tumor cells. However, E1A-RB mutations alone are not sufficient to prevent viral replication in normal cells. The addition of ‘tumor specific promoters’ improves tumor selectivity but drastically impacts replication and lytic activity. Therefore, a critical unmet need is the engineering of E2F addicted OVs that replicate like wildtype viruses in tumor cells, but leave normal cells unharmed. Methods: We have developed a proprietary platform that enables Ad therapeutics to be assembled from libraries of functional genomic parts that confer desirable properties. Here we describe E1 and E4 genomic modules that confer E2F dependent tumor selective replication. We assembled a panel of ̃50 viruses with combinations of mutations that ablate i) E1A interactions with RB/p107/p130 and/or EP300 ii) E4-ORF1 activation of PI3-Kinase and/or MYC iii) E4-ORF6/7 induced E2F1/DP1 dimerization. These viruses were screened in panels of primary, tumor, and CRISPR-engineered cells. Results: These studies reveal that E4-ORF6/7 functions independently of E1A to activate E2F transcriptional targets, S phase entry and viral replication. We show that viruses with both E1A and E4-ORF6/7 mutations (AdSyn-181) have the ideal tumor selectivity/efficacy profiles and outperform existing OVs. We also show that the deletion of p16 in primary cells rescues AdSyn-181 replication by ̃15 fold (̃20% wildtype virus), demonstrating the E2F dependence and tumor selectivity mechanism. Furthermore, we show that in tumor cells, which have multiple p16-CDK-RB pathway mutations, AdSyn-181 replication is rescued to 100% wildtype virus levels. ICVB-1042, which incorporates these selectivity mutations – together with other modules that enhance lytic cell death and spread, tumor tropisms and IV delivery – is in IND-enabling studies. Conclusions: Leveraging extensive research into the overlapping logic of viral and cancer pathways, we have engineered OVs, including ICVB-1042, that distinguish between tumor and normal cell proliferation. Furthermore, unlike therapies that target a single component, ICVB-1042 targets tumors with different p16-CDK-RB mutations, which all converge in activating oncogenic E2F transcription, driving exponential and tumoricidal viral replication.

Identification of RUNX1T1 as a potential epigenetic modifier in small-cell lung cancer.

Small-cell lung cancer (SCLC) can be subgrouped into common 'pure' and rare 'combined' SCLC (c-SCLC). c-SCLC features a mixed tumor histology of both SCLC and non-small-cell lung cancer (NSCLC). We performed targeted exome sequencing on 90 patients with SCLC, including two with c-SCLC, and discovered RUNX1T1 amplification specific to small cell tumors of both patients with c-SCLC, but in only 2 of 88 'pure' SCLC patients. RUNX1T1 was first identified in the fusion transcript AML1/ETO, which occurs in 12%-15% of acute myelogenous leukemia (AML). We further show higher expression of RUNX1T1 in the SCLC component of another c-SCLC tumor by in situ hybridization. RUNX1T1 expression was enriched in SCLC compared with all other cancers, including NSCLC, in both cell lines and tumor specimens, as shown by mRNA level and western blotting. Transcriptomic analysis of hallmark genes decreased by stable RUNX1T1 overexpression revealed a significant change in E2F targets. Validation experiments in multiple lung cancer cell lines showed that RUNX1T1 overexpression consistently decreased CDKN1A (p21) expression and increased E2F transcriptional activity, which is commonly altered in SCLC. Chromatin immunoprecipitation (ChIP) in these overexpressing cells demonstrated that RUNX1T1 interacts with the CDKN1A (p21) promoter region, which displayed parallel reductions in histone 3 acetylation. Furthermore, reduced p21 expression could be dramatically restored by HDAC inhibition using Trichostatin A. Reanalysis of ChIP-seq data in Kasumi-1 AML cells showed that knockdown of the RUNX1T1 fusion protein was associated with increased global acetylation, including the CDKN1A (p21) promoter. Thus, our study identifies RUNX1T1 as a biomarker and potential epigenetic regulator of SCLC.

Glycogen synthase kinase-3β participates in acquired resistance to gemcitabine in pancreatic cancer.

Acquisition of resistance to gemcitabine is a challenging clinical and biological hallmark property of refractory pancreatic cancer. Here, we investigated whether glycogen synthase kinase (GSK)‐3β, an emerging therapeutic target in various cancer types, is mechanistically involved in acquired resistance to gemcitabine in human pancreatic cancer. This study included 3 gemcitabine‐sensitive BxPC‐3 cell‐derived clones (BxG30, BxG140, BxG400) that acquired stepwise resistance to gemcitabine and overexpressed ribonucleotide reductase (RR)M1. Treatment with GSK3β‐specific inhibitor alone attenuated the viability and proliferation of the gemcitabine‐resistant clones, while synergistically enhancing the efficacy of gemcitabine against these clones and their xenograft tumors in rodents. The gemcitabine‐resensitizing effect of GSK3β inhibition was associated with decreased expression of RRM1, reduced phosphorylation of Rb protein, and restored binding of Rb to the E2 transcription factor (E2F)1. This was followed by decreased E2F1 transcriptional activity, which ultimately suppressed the expression of E2F1 transcriptional targets including RRM1, CCND1 encoding cyclin D1, thymidylate synthase, and thymidine kinase 1. These results suggested that GSK3β participates in the acquisition of gemcitabine resistance by pancreatic cancer cells via impairment of the functional interaction between Rb tumor suppressor protein and E2F1 pro‐oncogenic transcription factor, thereby highlighting GSK3β as a promising target in refractory pancreatic cancer. By providing insight into the molecular mechanism of gemcitabine resistance, this study identified a potentially novel strategy for pancreatic cancer chemotherapy.

Abstract 1279: SUMOylation upregulates EZH2 expression by modifying transcriptional factor E2F1

Abstract Enhancer of Zeste Homolog 2 (EZH2) is the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), which modifies histone H3 lysine 27 trimethylation (H3K27me3) at target promoters for gene silencing. High level of EZH2 expression is often observed in cancers, however the regulation of EZH2 expression is not well understood. We conducted genome wide RNA profiling in colorectal cancer cell line HCT116 with inducible knockdown of SUMO activating enzyme subunit 2 (SAE2). RNA-sequencing results showed EZH2 mRNA decreased and GSEA analysis indicated suppression of downstream genes of H3K27me3 upon SAE2 knockdown. Real-time PCR and western blot confirmed that knockdown of SAE2 decreased EZH2 mRNA and protein level as well as H3K27me3. The same effects were observed in breast cancer cell lines MB-MDA-231 and HCC1937. EZH2 promoter reporter assay demonstrated that SUMOylation enhanced EZH2 promoter transcriptional activity. SiRNA knockdown of SUMO E1 enzyme SAE2 or SUMO E2 enzyme UBC9 resulted in induction of CDH1, an target gene silenced by EZH2. EZH2 promoter luciferase reporter assay identified E2F transcription factor 1 (E2F1) as a major transcriptional activator of EZH2. The enhancement of EZH2 promoter transcriptional activity of E2F1 was induced by co-expression of SAE2 or SUMO1 but suppressed by expression of deSUMOylation enzyme SENP1. E2F1 SUMOylation site mutation (K266R) significantly decreased its transcriptional activation as measured by the EZH2 promoter reporter assay. SAE2 knockdown decreased the occupancy of E2F1 on EZH2 promoter as measured by Chromatin immunoprecipitation (ChIP) assay. The binding of E2F1 to EZH2 promoter region was significantly decreased when E2F1 SUMOyaltion site was mutated, which suggests that SUMOylation is required for E2F1 binding to EZH2 promoter. Western blot of tumor tissues from xenograft mouse model generated by colorectal cancer cell line HCT116 showed consistent results that knockdown of SAE2 decreased EZH2 mRNA and protein level in vivo. EZH2 protein level was also reduced in patient-derived xenograft (PDX) primary colorectal cancer cells when SAE2 was knockdown by siRNA transfection. Patient specimen data analysis showed SAE2 expression is associated with human colon and breast cancer patient survival and there is a strong positive correlation between EZH2 and SAE2 expression in tumor specimen from colon cancer and breast cancer patients. This data from patient samples supports our findings made in cellular and animal models. Of note, EZH2 and its partner SUZ12 are substrates of SUMOylation. However, their SUMOylation does not appear to impact the functions of PRC2. In conclusion, our results demonstrate that SUMOylation up-regulates EZH2 expression through regulating its gene expression via E2F1's transcriptional activity. Our findings reveal a novel mechanism of EZH2 regulation, indicating that SUMO E1 inhibitor could be synergistic with EZH2 inhibitor in colon and breast cancer therapy. Citation Format: Li Du, Yuan Chen, Steven Rosen. SUMOylation upregulates EZH2 expression by modifying transcriptional factor E2F1 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1279.

Differential expression of lung adenocarcinoma transcriptome with signature of tobacco exposure

Smoking accounts for almost 80–90% of lung cancer cases, which is also the most frequent cause of cancer-related deaths in humans. With over 60 carcinogens in tobacco smoke, cells dividing at the time of carcinogen exposure are at particular risk of neoplasia. The present study aimed to investigate global gene expression differences in lung adenocarcinoma (LUAD) tumour samples of current smokers and non-smokers, in an attempt to elucidate biological mechanisms underlying divergent smoking effects. Current and non-smoker tumour samples were analysed using bioinformatics tools, examining differences in molecular drivers of cancer initiation and progression, as well as evaluating the effect of smoking and sex on epithelial mesenchymal transition (EMT). As a result, we identified 1150 differentially expressed genes showing visible differences in the expression profiles between the smoking subgroups. The genes were primarily involved in cell cycle, DNA replication, DNA repair, VEGF, GnRH, ErbB and T cell receptor signalling pathways. Our results show that smoking clearly affected E2F transcriptional activity and DNA repair pathways including mismatch repair, base excision repair and homologous recombination. We observed that sex could modify the effects of PLA2G2A and PRG4 in LUAD tumour samples, whereas sex and smoking status might possibly have a biological effect on the EMT-related genes: HEY2, OLFM1, SFRP1 and STRAP. We also identified potential epigenetic changes smoking solely might have on EMT-related genes, which may serve as potential diagnostic and prognostic biomarkers for LUAD patients.

E2F1 sumoylation as a protective cellular mechanism in oxidative stress response

Oxidative stress is a ubiquitous threat to all aerobic organisms and has been implicated in numerous pathological conditions such as cancer. Here we demonstrate a pivotal role for E2F1, a cell cycle regulatory transcription factor, in cell tolerance of oxidative stress. Cells lacking E2F1 are hypersensitive to oxidative stress due to the defects in cell cycle arrest. Oxidative stress inhibits E2F1 transcriptional activity, independent of changes in association with Rb and without decreasing its DNA-binding activity. Upon oxidative insult, SUMO2 is extensively conjugated to E2F1 mainly at lysine 266 residue, which specifically modulates E2F1 transcriptional activity to enhance cell cycle arrest for cell survival. We identify SENP3, a desumoylating enzyme, as an E2F1-interacting partner. Oxidative stress inhibits the interaction between E2F1 and SENP3, which leads to accumulation of sumoylated E2F1. SENP3-deficient cells exhibit hypersumoylation of E2F1 and are resistant to oxidative insult. High levels of SENP3 in breast cancer are associated with elevated levels of E2F targets, high tumor grade, and poor survival. Given the prevalence of elevated levels of SENP3 across numerous cancer types, the SENP3-E2F1 axis may serve as an avenue for therapeutic intervention in cancer.

Role of E2F3 and shugoshin I in epithelial-to-mesenchymal transition and cell invasion in breast cancer.

e13100 Background: Triple-negative breast cancer (TNBC) is the most aggressive and poorly prognostic breast cancer subtype, yet there are currently no biological therapies against this subtype. Our laboratory is finding the sources of novel biological targets in TNBC by studying the E2F transcription factors, which are essential for cellular proliferation and maintenance of genomic stability. While the deregulated Rb/E2F pathway signals the epithelial-to-mesenchymal transition (EMT), the underlining mechanism of how E2Fs drive EMT in TNBC remains unknown. We recently published that the E2F transcriptional activators (E2Fs) are overexpressed in the vast majority of TNBC and that their overexpression upregulates mitotic kinases such as TTK, which we have shown to induce EMT and invasion in TNBC cells. We also demonstrated that the E2Fs maintain genomic integrity in part through Shugoshin I (SGO1), which normally controls chromosome cohesion; however, the role of SGO1 in EMT in breast cancer is unknown. Our hypothesis is that E2F3 and SGO1 are highly expressed in TNBC and that their overexpression modulates EMT genes, thus promoting cell invasion. Methods: To test our hypothesis, we conducted siRNA transfection to knockdown E2F3 and SGO1 in MDA-MB-231 and Hs578t, which are TNBC cells. After 48 hours, we evaluated mRNA levels of EMT-related genes after E2F3 or SGO1 depletion using RT-PCR analysis. We also evaluated the effects of SGO1 depletion in protein localization by immunofluorescence. Furthermore, we evaluated the invasive behavior of MDA-MB-231 and Hs578t cells after SGO1 depletion using a Boyden Chamber Assay. Results: Our results demonstrate that E2F3 and SGO1 depletion decrease MMP3 mRNA levels. Moreover, E2F3 and SGO1 depletion restore E-cadherin expression and localization. Furthermore, E2F3 and SGO1 depletion significantly reduce cell invasion in MDA-MB-231 and Hs578t cells. Conclusions: Our results suggest that SGO1 is a promising drug target for breast cancer metastasis since EMT and invasion are essential early steps in breast cancer metastasis and E2F3 is presently undruggable.

A Novel Rhynchophylline Analog, Y396, Inhibits Endothelial Dysfunction Induced by Oxidative Stress in Diabetes Through Epidermal Growth Factor Receptor.

Aims: Endothelial dysfunction appears in early diabetes mellitus partially because of epidermal growth factor receptor (EGFR) abnormal activation and downstream oxidative stress. The aim of this study was to determine whether Y396, a synthesized analog of rhynchophylline, could protect against endothelial dysfunction in diabetes and the underlying molecular mechanism. Results: Y396 could directly target the EGFR and inhibit its phosphorylation induced by high glucose and EGF, downstream translocation to the nucleus of E2F1, and its transcriptional activity and expression of Nox4. Diabetes-induced endothelium malfunction was ameliorated by Y396 treatment through EGFR inhibition. Downstream oxidative stress was decreased by Y396 in the aortas of type 1 diabetes mellitus mice and primary rat aorta endothelial cells (RAECs). Y396 could also ameliorate tunicamycin-induced oxidative stress in the aorta and RAECs. In addition, we again determined the protective effects of Y396 on high-fat diet/streptozotocin-induced type 2 diabetes mellitus. Innovation: This is the first study to demonstrate that Y396, a novel rhynchophylline analog, suppressed high-glucose-induced endothelial malfunction both in vivo and in vitro by inhibiting abnormal phosphorylation of EGFR. Our work uncovered EGFR as a novel therapeutic target and Y396 as a potential therapy against diabetes-induced complication. Conclusion: Y396 could directly bind with EGFR, and inhibit its phosphorylation and downstream E2F1 transcriptional activity. It could also preserve tunicamycin-evoked endothelial dysfunction and oxidative stress. It could protect against diabetes-induced endothelium malfunction in vivo through EGFR inhibition and downstream oxidative stress. Antioxid. Redox Signal. 32, 743-765.

Abstract P6-07-06: Shugoshin I signals epithelial-mesenchymal transition and promotes cell invasion in breast cancer

Abstract Despite being the most aggressive and poorly prognostic breast cancer subtype, there are currently no biological therapies against triple-negative (TN, or ER-PR-Her2-) breast cancers. Our laboratory is finding the sources of novel biological targets in TNBC by studying the E2F transcription factors, which are essential for cellular proliferation and maintenance of genomic stability. We recently published that the E2F transcriptional activators are overexpressed in the vast majority of TNBC and can dictate the high percentage of mitotic cells in mammary tumors. We also demonstrated that that the E2Fs control multiple mitotic regulators and maintain genomic integrity through Shugoshin I (SGO1) and BubR1. SGO1 is essential for chromosome segregation by preventing premature dissociation of the cohesion complex and sister chromatids after prophase. Until recently, SGO1 research was restricted to embryogenesis; however, recent studies have shown a role for SGO1 in tumorigenesis and epithelial-mesenchymal transition (EMT), but none have been done in breast cancer. Thus, we investigated SGO1 expression and genetic alteration in open databases such as KM-Plotter and cBioPortal and found that SGO1 is highly expressed in triple-negative breast cancer subtype and that its expression correlates with poor prognostic factors. Therefore, we decided to investigate the role of SGO1 in breast cancer EMT and cell invasion. Our hypothesis is that SGO1 modulates EMT genes, thus promoting cell invasion. To test our hypothesis, we conducted siRNA transfection to knockdown SGO1 in MDA-MB-231 and Hs578t, which are TNBC cells. After 48 hours, we evaluated mRNA levels of EMT-related genes after SGO1 depletion using RT-PCR analysis. We also evaluated the effects of SGO1 depletion in protein localization by immunofluorescence analysis. Furthermore, we evaluated the invasive behavior of MDA-MB-231 and Hs578t cells after SGO1 depletion using a Boyden Chamber Assay. Our results demonstrate that SGO1 depletion signals EMT by decreasing N-cadherin and increasing E-cadherin mRNA levels. SGO1 depletion also restores protein expression of E-cadherin in MDA-MB-231 cells. Strikingly, SGO1 depletion significantly reduces invasion in MDA-MB-231 and Hs578t cells. Our results suggest that SGO1 is a promising drug target for breast cancer metastasis since EMT and invasion are essential early steps in breast cancer metastasis. Currently, we are evaluating SGO1 expression in tissue microarrays derived from breast cancer patients (including Luminal and TNBC subtypes), in order to determine the percentage of those patients expressing Sgo1, as well as to establish important correlations with surrogate markers of prognosis (EMT, proliferation, mitosis). Our results may help establish new druggable targets in order to prevent and reduce metastasis in TNBC patients. Citation Format: Shirley Jusino, Harold I. Saavedra. Shugoshin I signals epithelial-mesenchymal transition and promotes cell invasion in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-07-06.

ILF2 cooperates with E2F1 to maintain mitochondrial homeostasis and promote small cell lung cancer progression.

ObjectiveMitochondria play multifunctional roles in carcinogenesis. Deciphering uncertainties of molecular interactions within mitochondria will promote further understanding of cancer. Interleukin enhancer binding factor 2 (ILF2) is upregulated in several malignancies, however, much remains unknown regarding ILF2 in small cell lung cancer (SCLC). In the current study, we explored ILF2’s role in SCLC and demonstrated its importance in mitochondria quality control.MethodsColony formation, cell proliferation, cell viability and xenograft studies were performed to examine ILF2’s role on SCLC progression. Glucose uptake, lactate production, cellular oxygen consumption rate and extracellular acidification rate were measured to examine the effect of ILF2 on glucose metabolism. RNA-sequencing was utilized to explore genes regulated by ILF2. E2F1 transcriptional activity was determined by dual luciferase reporter assay. Mitochondria quantification and mitochondrial membrane potential assays were performed to examine mitochondrial quality. Gene expression was determined by RT-qPCR, Western blot and IHC assay.ResultsILF2 promotes SCLC tumor growth in vitro and in vivo. ILF2 elevates oxidative phosphorylation expression and declines glucose intake and lactate production. Genome-wide analysis of ILF2 targets identified a cohort of genes regulated by E2F1. In consistent with this, we found ILF2 interacts with E2F1 in SCLC cells. Further studies demonstrated that suppression of E2F1 expression could reverse ILF2-induced tumor growth and enhanced mitochondria function. Significantly, expression of ILF2 is progressively increased during SCLC progression and high ILF2 expression is correlated with higher histologic grades, which indicates ILF2’s oncogenic role in SCLC. ConclusionsOur results demonstrate that ILF2 interacts with E2F1 to maintain mitochondria quality and confers SCLC cells growth advantage in tumorigenesis.

Ribosomal protein uL3 targets E2F1 and Cyclin D1 in cancer cell response to nucleolar stress

Several experimental strategies in the treatment of cancer include drug alteration of cell cycle regulatory pathways as a useful strategy. Extra-ribosomal functions of human ribosomal protein L3 (uL3) may affect DNA repair, cell cycle arrest and apoptosis. In the present study, we demonstrated that uL3 is required for the activation of G1/S transition genes. Luciferase assays established that uL3 negatively regulates the activity of E2F1 promoter. Induced ribosome-free uL3 reduces Cyclin D1 mRNA and protein levels. Using protein/protein immunoprecipitation methods, we demonstrated that uL3 physically interacts with PARP-1 affecting E2F1 transcriptional activity. Our findings led to the identification of a new pathway mediated by uL3 involving E2F1 and Cyclin D1 in the regulation of cell cycle progression.

PF561 DUAL‐TARGETING OF PROMOTER AND ENHANCER DRIVEN PROCESSES SUPPRESSES MYELOMA CELL GROWTH AND VIABILITY VIA PERTURBATION OF THE MYELOMA PROLIFERATIVE PROGRAM

Background:Cancer cells are characterized by perturbed transcriptional profiles. These dysregulated programs create dependencies on transcriptional regulators not predicted by genetic changes. Tumor cells, including multiple myeloma cells, tend to develop striking dependencies on super‐enhancer regulatory elements. In addition to super‐enhancers, promoter proximal transcription factors such as E2F1 and SP1 play a prominent role in myeloma cell biology via their control of proliferative and survival genes. Importantly, our genome‐wide profiling of the MM epigenome landscape revealed that promoter‐ and enhancer‐associated factors govern distinct biological functions providing a non‐overlapping control of the myeloma transcriptome.Aims:The goal of this study is to improve our understanding of global gene regulation in MM, with the aim of deploying novel therapeutics to target uncontrolled MM cell proliferation.Methods:We performed integrated genomic analysis using a combination of ChIP‐seq, ATAC‐seq, and RNA‐seq technologies as well as loss of function screening in MM cell lines and primary MM cells.Results:Global chromatin analysis of MM cells revealed two distinct regulatory axes, with SP1, DP1, E2F and MYC predominantly localized to promoters of growth/proliferation genes and CDK9, BETs and other factors disproportionately enriched at enhancer‐regulated tissue‐specific genes. This divergent enhancer and promoter axes is also observed in diffuse large B‐cell lymphoma, suggesting a broader transcriptome control process. Importantly, we have observed that dual inhibition of promoter and enhancer axes using RNA interference shows a superior inhibition of MM cell growth and viability both in vitro and in vivo compared to single perturbation alone, providing an important molecular mechanism for combination therapy. We have further gone on to target TFs SP1 with a small molecule inhibitor Terameprocol which inhibits its DNA binding activity and a cell‐permeable peptide to disrupt the heterodimerization of E2F1 with its partner DP1 responsible for the E2F transcriptional activity. These strategies have been successful in inhibiting MM cell growth and viability, and the effect was augmented in the presence of inhibitors of enhancers, such as the small molecule BRD4 inhibitor JQ1.We have further investigated inhibitors of upstream regulators of the pRB‐E2F axis such as CDK4/6 to impact E2F1 activity. CDK4–6 complexes can phosphorylate RB, releasing the E2F and modulating the expression of E2F target genes. Therefore, we investigated the combination of CDK4/6 inhibitor Palbociclib with low doses of JQ1 and observed a profound effects on E2F promoter driven transcriptional activity, and a highly synergistic effect on growth and survival in a large panel of cell lines and primary MM cells from newly diagnosed and relapsed patients. Cell cycle analysis revealed complete G1 arrest after treatment. Importantly, the combination regimen had no significant effect on healthy donor PBMCs activated with PHA, suggesting a favorable therapeutic index.Summary/Conclusion:These data implicate the existence of a sequestered cellular function by promoter and enhancer driven processes providing non‐overlapping vulnerabilities. Simultaneous targeting of these processes using clinically applicable agents can synergistically impair the myeloma proliferative program, with potential for development of a promising therapeutic strategy.