Expression Of Tumor Suppressor Genes Research Articles

Elucidating the direct effects of the novel HDAC inhibitor bocodepsin (OKI-179) on T cells to rationally design regimens for combining with immunotherapy.

Histone deacetylase inhibitors (HDACi) are currently being explored for the treatment of both solid and hematological malignancies. Although originally thought to exert cytotoxic responses through tumor-intrinsic mechanisms by increasing expression of tumor suppressor genes, several studies have demonstrated that therapeutic responses depend on an intact adaptive immune system: particularly CD8 T cells. It is therefore critical to understand how HDACi directly affects T cells in order to rationally design regimens for combining with immunotherapy. In this study, we evaluated T cell responses to a novel class-selective HDACi (OKI-179, bocodepsin) by assessing histone acetylation levels, which revealed rapid responsiveness accompanied by an increase in CD4 and CD8 T cell frequencies in the blood. However, these rapid responses were transient, as histone acetylation and frequencies waned within 24 hours. This contrasts with in vitro models where high acetylation was sustained and continuous exposure to HDACi suppressed cytokine production. In vivo comparisons demonstrated that stopping OKI-179 treatment during PD-1 blockade was superior to continuous treatment. These findings provide novel insight into the direct effects of HDAC inhibitors on T cells and that treatment schedules that take into account acute T cell effects should be considered when combined with immunotherapies in order to fully harness the tumor-specific T cell responses in patients.

Anticancer Potential of Flavonoids: An Overview with an Emphasis on Tangeretin.

Natural compounds with pharmacological activity, flavonoids have been the subject of an exponential increase in studies in the field of scientific research focused on therapeutic purposes due to their bioactive properties, such as antioxidant, anti-inflammatory, anti-aging, antibacterial, antiviral, neuroprotective, radioprotective, and antitumor activities. The biological potential of flavonoids, added to their bioavailability, cost-effectiveness, and minimal side effects, direct them as promising cytotoxic anticancer compounds in the optimization of therapies and the search for new drugs in the treatment of cancer, since some extensively antineoplastic therapeutic approaches have become less effective due to tumor resistance to drugs commonly used in chemotherapy. In this review, we emphasize the antitumor properties of tangeretin, a flavonoid found in citrus fruits that has shown activity against some hallmarks of cancer in several types of cancerous cell lines, such as antiproliferative, apoptotic, anti-inflammatory, anti-metastatic, anti-angiogenic, antioxidant, regulatory expression of tumor-suppressor genes, and epigenetic modulation.

Exploring the Role of microRNAs in Glioma Progression, Prognosis, and Therapeutic Strategies.

Gliomas, which arise from glial cells in the brain, remain a significant challenge due to their location and resistance to traditional treatments. Despite research efforts and advancements in healthcare, the incidence of gliomas has risen dramatically over the past two decades. The dysregulation of microRNAs (miRNAs) has prompted the creation of therapeutic agents that specially target them. However, it has been reported that they are involved in complex signaling pathways that contribute to the loss of expression of tumor suppressor genes and the upregulation of the expression of oncogenes. In addition, numerous miRNAs promote the development, progression, and recurrence of gliomas by targeting crucial proteins and enzymes involved in metabolic pathways such as glycolysis and oxidative phosphorylation. However, the complex interplay among these pathways along with other obstacles hinders the ability to apply miRNA targeting in clinical practice. This highlights the importance of identifying specific miRNAs to be targeted for therapy and having a complete understanding of the diverse pathways they are involved in. Therefore, the aim of this review is to provide an overview of the role of miRNAs in the progression and prognosis of gliomas, emphasizing the different pathways involved and identifying potential therapeutic targets.

Urolithin B inhibits proliferation and migration and promotes apoptosis and necrosis by inducing G2/M arrest and targeting MMP-2/-9 expression in osteosarcoma cells.

Osteosarcoma (OS) is the most prevalent primary bone cancer, with a high morbidity and mortality rate. Over the past decades, therapeutic approaches have not considerably improved patients' survival rates, and further research is required to find efficient treatments for OS. Data from several studies have shown that urolithin B (UB), the intestinal metabolite of polyphenolic ellagitannins, is emerging as a new class of anticancer compounds, yet its effect on OS cancer cells remains elusive. Herein, we investigated UB's antimetastatic, antiproliferative, and apoptotic effects on the MG-63 OS cell line. Cell viability assay, annexin V/propidium iodide staining, cell cycle arrest analysis, determination of the gene expression of MMP-2, MMP-9, Bax, Bcl-2, and p53 messenger RNA (mRNA), evaluation of reactive oxygen species (ROS) generation and migration, and MMP-2 and MMP-9 protein expression assessments were performed. UB caused late apoptosis, necrosis, G2/M arrest, and ROS generation in MG-63 cells. It increased the mRNA expression of the p53 tumor suppressor and Bax proapoptotic genes. UB also inhibited the migration and metastatic behavior of MG-63 OS cells by downregulating mRNA and MMP-2 and MMP-9 protein expression. In general, although further in vivo investigations are warranted, the current results showed that UB might be utilized as a potential novel natural compound for OS therapy due to its nontoxic, antiproliferative, and antimetastatic nature.

Impact of tumor suppressor genes inactivation on the multidrug resistance phenotype of hepatocellular carcinoma cells

The response of advanced hepatocellular carcinoma (HCC) to pharmacological treatments is unsatisfactory and heterogeneous. Inactivation of tumor suppressor genes (TSGs) by genetic and epigenetic events is frequent in HCC. This study aimed at investigating the impact of frequently altered TSGs on HCC chemoresistance. TSG alterations were screened by in silico analysis of TCGA-LIHC database, and their relationship with survival was investigated. These TSGs were silenced in HCC-derived cell lines using CRISPR/Cas9. TLDA was used to determine the expression of a panel of 94 genes involved in the resistome. Drug sensitivity, cell proliferation, colony formation and cell migration were assessed. The in silico study revealed the down-regulation of frequently inactivated TSGs in HCC (ARID1A, PTEN, CDH1, and the target of p53, CDKN1A). The presence of TP53 and ARID1A variants and the low expression of PTEN and CDH1 correlated with a worse prognosis of HCC patients. In PLC/PRF/5 cells, ARID1A knockout (ARID1AKO) induced increased sensitivity to cisplatin, doxorubicin, and cabozantinib, without affecting other characteristics of malignancy. PTENKO and E-CadKO showed minimal changes in malignancy, resistome, and drug response. In p53KO HepG2 cells, enhanced malignant properties and higher resistance to cisplatin, doxorubicin, sorafenib, and regorafenib were found. This was associated with changes in the resistome. In conclusion, the altered expression and function of several TSGs are involved in the heterogeneity of HCC chemoresistance and other features of malignancy, contributing to the poor prognosis of these patients. Individual identification of pharmacological vulnerabilities is required to select the most appropriate treatment for each patient.

Synthetic Reader-Actuators Targeted to Polycomb-Silenced Genes Block Triple-Negative Breast Cancer Proliferation and Invasion.

Scientists have used pharmacological inhibitors of polycomb proteins to restore the expression of tumor suppressor genes and stop cancer proliferation and invasion. A major limitation of this approach is that key transcriptional activators, such as TP53 and BAF SWI/SNF, are often mutated in cancer. Poor clinical results for polycomb-targeting therapies in solid cancers, including triple-negative breast cancer (TNBC), could discourage the further development of epigenetic monotherapies. Here, we performed epigenome actuation with a synthetic reader-actuator (SRA) that binds trimethylated histone H3 lysine 27 in polycomb chromatin and modulates core transcriptional activators. In SRA-expressing TNBC BT-549 cells, 122 genes become upregulated ≥2-fold, including the genes involved in cell death, cell cycle arrest, and migration inhibition. The SRA-expressing spheroids showed reduced size in Matrigel and loss of invasion. Therefore, targeting Mediator-recruiting regulators to silenced chromatin can activate tumor suppressors and stimulate anti-cancer phenotypes, and further development of robust gene regulators might benefit TNBC patients.

Novel quinoxaline-based VEGFR-2 inhibitors to halt angiogenesis

Vascular endothelial growth factor receptor-2 is a dynamic target for therapeutic intervention in various types of cancer. This study was aimed at exploring the VEGFR-2 inhibitory activity of a novel library of quinoxalin-2-one derivatives such as 3-furoquinoxaline carboxamides, 3-pyrazolylquinoxalines, and 3-pyridopyrimidyl-quinoxalines. Among them, 6c, 7a, and 7d-f produced remarkable cytotoxicity against HCT-116 (IC50′s 4.28–9.31 µM) and MCF-7 (IC50′s 3.57–7.57 µM) cell lines using the MTT assay and doxorubicin (DOX) as a reference standard. Interestingly, results of cytotoxicity towards the human fibroblast cell line WI38 revealed that these hits demonstrated higher selectivity indices towards both HCT-116 (SI 8.69–23.19) and MCF-7 (SI 9.48–27.80) than DOX, SI 0.72 and 0.90, respectively. Then, these hits were subjected to a mechanistic study; they showed direct inhibition of VEGFR-2. Impressively, compound 7f displayed 1.2 times the VEGFR-2 inhibitory activity of sorafenib. The antiangiogenic potential of 7f was proved via lowering the level of VEGF-A, than that of control. It as well, exhibited scratch closure percent of 61.8%, compared with 74.5% of control at 48 hrs, indicating the potential anti-migratory effect of the compound 7f. It significantly increased the expression of tumor suppressor gene (p53) on MCF-7 cells by almost 18 folds and upregulated the caspase-3 level by 10.7 folds, compared to the control. Cell cycle analysis revealed cell cycle arrest at G2/M together with a PreG increase which indicated apoptosis induction potential. Annexin V-FITC apoptosis results proposed the two modes of cell death (apoptosis and necrosis) as an inherent mechanism of cytotoxicity of compound 7f. Molecular docking further supported the mechanism showing the affinity of target compounds for VEGFR-2 active site. Moreover, physicochemical and drug-like properties were assessed from the ADME properties.

Prognosis of colon cancer patients based on enhancer RNAs-related genes.

Colon cancer (CC) is a cancer of the large intestine with high prevalence and poor prognosis. enhancer RNAs. Therefore, valuable tools or biomarkers for predicting patient status, directing clinical practice, and reducing overtreatment are needed. Enhancer RNAs (eRNAs), a class of noncoding RNAs transcribed from enhancers, have been shown to function as regulators of oncogene or tumor suppressor gene expression. The aim of our study was to explore the potential roles of eRNAs and their target enhancer-related genes (ERGs) in the prognosis of CC. Selected CC cases (stage I-III) from The Cancer Genome Atlas database were used as a training set, and cases from the Gene Expression Omnibus were used as the validation set. ERGs associated with prognosis were screened through three steps: potential, candidate, and prognosis ERGs. Multivariate Cox proportional hazards analysis was used to identify independent prognostic factors, and a nomogram was created. Calibration curves were drawn by comparing predicted and observed survival probability. For validation, the calibration curves and ROC analysis were also applied to two external validation sets. The biological significance and clinical application of the genes obtained were investigated. Based on the multiple tiers of strict screening, 11 prognostic ERGs were obtained, which were combined to obtain a prognosis signature. A compound nomogram integrating age, TNM classification, and the prognostic signature was constructed. The model was reliable in distinguishing the risk of patients with stage I-III CC, with AUCs of 0.78 and 0.70 at 5 and 7years, respectively. There was good reproducibility in calibration curves. The prognostic model also yielded good prediction capability in the validation sets. In this study, the usefulness and specificity of the ERGs in prognosis were described, which should be considered a key feature in the clinical guidance of CC patients with early stage. We concluded that the major implications of the eRNAs and ERGs should be valued, which would be an emerging hallmark in the prognosis of cancer.

Appraisals on the anticancer properties of Mentha species using bioassays and docking studies

The medicinal properties of Mentha species are highly regarded due to their pro-health biological properties. This study aimed to investigate the fractions and bioactive compounds that are responsible for anticancer properties of the Mentha species. Firstly, the cytotoxic effects of leaves and hairy roots extracts from two Mentha species including Mentha piperita L. and Mentha spicata L., were assessed by MTT assay. Then, a bioassay-guided fractionation of an extract with higher toxicity against cancer cells was conducted to yield the bioactive compound. Among all the four extracts tested, the leaf extract of M. piperita possessed the greatest cytotoxic activity against A549, MCF-7, and PC-3 cells. Bioassay-guided fractionation revealed that the purified fraction showed a cytotoxic effect (IC50 =54.8 μg/mL) against the PC-3 cancer cell line and exhibited potent anticancer activity by inducing apoptosis and wound healing properties, as well as upregulating the expression of tumor suppressor genes. Analytical investigations on the purified fraction utilizing LC-MS NMR, and FT-IR methodologies indicated that the identified anticancer metabolite was considered as a furanic compound called 2-ethylhexyl 5-hydroxyfuran-2 carboxylate, which is reported for the first time here. Molecular docking analyses indicated that the furan derivative has a great potential to differentially interact with cancer-related receptors. Therefore, this evidence suggests that the Mentha species has valuable anticancer bioactive compounds that could be considered in phytochemical-based cancer therapy programs.

Efficacy of Prunus armenica, Bee venom, and their combinations on p53 and Bcl-2 gene expression in human pancreatic and lung cancer cells

Malignancies are one of the world’s most serious health problems. Pancreatic and lung cancers are two of the most lethal types of cancer, with 5-year survival rates ranging from 10 to 20%. Despite decades of investigation, the molecular processes and selectivity of Prunus armenica and Bee venom as anticancer drugs are still largely un-understood. The goal of our study is to determine the expression levels ofp53 and Bcl-2 mRNAs obtained from human pancreatic (PANC-1) and lung (A549) cancer cells treated with Prunus armenica, Bee venom or their combinations by using MTT and real time PCR assays. We showed that in a concentration-dependent manner of Prunus armeniaca, bee venom, and a combination of both, the cell growth was significantly decreased. Our findings also revealed an adverse association between tumor suppressor (p53) and proto-oncogene (Bcl-2) gene expression in both cancer cells treated with diverse substances vs untreated. As a consequence, the tested compounds dramatically alter p53 and Bcl-2 gene expression, suggesting that they might be useful in the prevention or treatment of pancreatic and lung cancers.

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.

EPIC-0307-mediated selective disruption of PRADX-EZH2 interaction and enhancement of temozolomide sensitivity to glioblastoma via inhibiting DNA repair and MGMT.

Temozolomide (TMZ) treatment efficacy in glioblastoma (GBM) has been limited by resistance. The level of O-6-methylguanine-DNA methyltransferase (MGMT) and intrinsic DNA damage repair factors are important for the TMZ response in patients. Here, we reported a novel compound, called EPIC-0307, that increased TMZ sensitivity by inhibiting specific DNA damage repair proteins and MGMT expression. EPIC-0307 was derived by molecular docking screening. RNA immunoprecipitation (RIP), and chromatin immunoprecipitation by RNA (ChIRP) assays were used to verify the blocking effect. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) assays were performed to explore the mechanism of EPIC-0307. A series of in vivo and in vitro experiments were designed to evaluate the efficacy of EPIC-0307 in sensitizing GBM cells to TMZ. EPIC-0307 selectively disrupted the binding of PRADX to EZH2 and upregulated the expression of P21 and PUMA, leading to cell cycle arrest and apoptosis in GBM cells. EPIC-0307 exhibited a synergistic inhibitory effect on GBM when combined with TMZ by downregulating TMZ-induced DNA damage repair responses and epigenetically silencing MGMT expression through modulating the recruitment of ATF3-pSTAT3-HDAC1 regulatory complex to the MGMT promoter. EPIC-0307 demonstrated significant efficacy in suppressing the tumorigenesis of GBM cells, restoring TMZ sensitivity. This study identified a potential small-molecule inhibitor (SMI) EPIC-0307 that selectively disrupted the PRADX-EZH2 interaction to upregulate expressions of tumor suppressor genes, thereby exerting its antitumor effects on GBM cells. EPIC-0307 treatment also increased the chemotherapeutic efficacy of TMZ by epigenetically downregulating DNA repair-associate genes and MGMT expression in GBM cells.

TRNA-derived fragments (tRFs) as regulators of oncogenes and tumor suppressor genes in head and neck squamous cell carcinoma.

e18082 Background: Roughly 50,000 individuals are diagnosed with head and neck cancers in the United States yearly. Though risk factors and common therapeutic strategies are well-established, early detection of head and neck cancers remains challenging. One strategy for early detection is high-throughput analysis of both coding and noncoding RNA transcripts, which has become increasing implicated in cancer pathogenesis. One such noncoding transcript is tRNA-derived fragments (tRFs), which are the products of enzymatic cleavage of precursor tRNAs. Though tRFs have been suggested to play important roles in the regulation of mRNA translation (similar to other noncoding RNAs), their utility as cancer biomarkers is not well-known. Methods: In this study, we investigate tRFs for correlation to cancer prevalence with specific consideration of translational regulation of oncogenes (OG) and tumor suppressor genes (TSG). tRF read counts were obtained for 499 head and neck squamous cell carcinoma tumor tissue samples and 44 adjacent normal samples. Results: The read counts between both groups were compared to identify tRFs dysregulated in tumor samples, which may be pertinent biomarkers for diagnosis. Ten tRFs were found to be significantly dysregulated in cancer samples. These tRFs were further assessed for correlation to expression of OGs and TSGs using the COSMIC database. Due to the mRNA-degrading nature of tRFs, we analyzed tRF abundance for inverse correlation to gene expression. Four tRFs upregulated in tumor samples showed significant anticorrelation to select TSG expression (ATP2B3, CSMD3, LZTR1, NTHL1, PTPRC), and three tRFs downregulated in tumor samples showed anticorrelation to OG expression (A1CF, MPL, SALL4, TNC). Notably, LeuCAG 5’ tRF was associate with high binding affinity for the TSGs CSMD3 and LZTR1, supporting the likelihood of tRF-mediated degradation. Interestingly, this tRF was inversely correlated with patient survival length. LeuCAG 5’ was also of significantly lesser abundance in patients who experienced complete or partial remission following chemotherapy treatment, further indicating this tRFs’ clinical relevance in HNSCC, potentially as an oncogenic-related agent. Conclusions: Investigation into these tRFs may reveal their potential for use in early diagnosis. High abundance of this tRF may be indicative of oncogenesis. In exploring tRFs’ for their potential as biomarkers of cancer, they may prove useful in allowing earlier diagnosis. Moreover, studying tRFs may provide additional considerations for patient care and treatment.

Novel sights on therapeutic, prognostic, and diagnostics aspects of non-coding RNAs in glioblastoma multiforme.

Glioblastoma Multiforme (GBM) is the primary brain tumor and accounts for 200,000 deaths each year worldwide. The standard therapy includes surgical resection followed by temozolomide (TMZ)-based chemotherapy and radiotherapy. The survival period of GBM patients is only 12-15months. Therefore, novel treatment modalities for GBM treatment are urgently needed. Mounting evidence reveals that non-coding RNAs (ncRNAs) were involved in regulating gene expression, the pathophysiology of GBM, and enhancing therapeutic outcomes. The combinatory use of ncRNAs, chemotherapeutic drugs, and tumor suppressor gene expression induction might provide an innovative, alternative therapeutic approach for managing GBM. Studies have highlighted the role of Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in prognosis and diagnosis. Dysregulation of ncRNAs is observed in virtually all tumor types, including GBMs. Studies have also indicated the blood-brain barrier (BBB) as a crucial factor that hinders chemotherapy. Although several nanoparticle-mediated drug deliveries were degrading effectively against GBM in vitro conditions. However, the potential to cross the BBB and optimum delivery of oligonucleotide RNA into GBM cells in the brain is currently under intense clinical trials. Despite several advances in molecular pathogenesis, GBM remains resistant to chemo and radiotherapy. Targeted therapies have less clinical benefit due to high genetic heterogeneity and activation of alternative pathways. Thus, identifying GBM-specific prognostic pathways, essential genes, and genomic aberrations provide several potential benefits as subtypes of GBM. Also, these approaches will provide insights into new strategies to overcome the heterogenous nature of GBM, which will eventually lead to successful therapeutic interventions toward precision medicine and precision oncology.

Mechanisms of Cancer-killing by Quercetin; A Review on Cell Death Mechanisms.

Cancer drug resistance has always been a serious issue regarding cancer research and therapy. Different cancers undergo different mutations, which may cause suppression of tumor suppressor genes, inhibition of apoptosis, stimulation of drug resistance mediators, and exhaustion of the immune system. The modulation of pro-death and survival-related mediators is an intriguing strategy for cancer therapy. Several nature-derived molecules, e.g., quercetin, have shown interesting properties against cancer through the modulation of apoptosis and autophagy mediators. Such molecules, e.g., quercetin, have been shown to stimulate apoptosis and other types of cell death pathways in cancers via the modulation of ROS metabolism. Quercetin may affect immune system function and trigger the expression and activity of tumor suppressor genes. Furthermore, it may suppress certain multidrug resistance mechanisms in cancer cells. This paper aims to review the effects of quercetin on various cell death mechanisms such as apoptosis, autophagic cell death, senescence, ferroptosis, and others.

Chemopreventive potential of kimchi, an ethnic food from Korea, against colorectal carcinogenesis associated with red meat intake

Kimchi, the traditional fermented vegetables with seasonings from Korea, is globally renowned as healthy food with anticancer properties. Colorectal cancer is the world’s third leading cancer with an increasing incidence rate over the past years. High red meat intake is strongly associated with colorectal cancer, and the World Health Organization has identified red meat as probably carcinogenic to humans (Group 2A). This study aimed to investigate the chemopreventive potential of cabbage kimchi against the promotion of colorectal cancer by red meat in vivo using carcinogen-injected rats. Rats were fed by AIN-76 diet enriched in freeze-dried beef sirloin supplemented or not with kimchi powder for 120 days prior to killing. Kimchi supplementation at moderate (5%) and high (10%) concentration significantly suppressed the formation of precancerous lesions in the colon of red meat-fed rats. Using fecal water, the aqueous extract of feces, we found out that kimchi supplementation limited iron-mediated oxidation and reduced lipid peroxidation in the feces of rats. Furthermore, kimchi supplementation reduced the toxicity of fecal water of red meat-fed rats toward human colonic epithelial cells in vitro by suppressing the formation of cellular reactive oxygen species (ROS). The chemopreventive effects of kimchi were associated with the acidification of fecal matrix and increase in fecal lactic acid bacteria (LAB). Gene expression analysis in the colon of rats demonstrated that kimchi supplementation prevented colorectal carcinogenesis by up-regulating the expression of tumor-suppressor genes and antioxidant enzymes, as well as by down-regulating the expression of proinflammatory proteins. Taken together, our findings suggested that kimchi consumption is correlated with lower promotion of colorectal cancer associated with red meat intake.

Abstract 6001: Epigenetic regulation of BARD1 confers resistance to anti-VEGF therapy in ovarian cancer

Abstract Introduction: Vascular endothelial growth factor (VEGF) is a key promoter of angiogenesis and disease progression in epithelial ovarian cancer (OC). Bevacizumab, a humanized anti-VEGF monoclonal antibody, is approved for treatment of patients with ovarian and other cancers. Despite the wide use of anti-VEGF therapy for cancer treatment, most patients develop resistant disease. Therefore, new combinations/strategies are needed to reduce adaptive resistance to anti-VEGF antibody (AVA) therapy. Our aim is to determine the potential role of epigenetic changes in AVA-adaptive resistance. We identified and characterized epigenetic changes in BARD1, a tumor suppressor, at the emergence of AVA resistance. Methods: To define epigenetic changes during AVA therapy induced resistance, we performed series of in vitro (methylation array, RNA sequencing) and in vivo (orthotopic OC (PDX-MDA-OVCA1, immunocompetent-IG10, and immunocompromised-SKOV3) and fibrosarcoma-HT1080, an inherently VEGF resistant model) experiments. The biological effects of DNA methylation agent (5`-Azacytidine; 5`-Aza) with B20 (murine anti-VEGF antibody) treatment were subsequently tested using in vivo models. Results: To determine whether 5`-Aza can enhance the efficacy of AVA therapy and overcome resistance to AVA in OC, we established AVA-resistant SKOV3 model. In AVA-resistant mice treated with B20 + 5`-Aza, there was a statistically significant reduction in tumor weight (p=0.03). Next, we performed a Human 450k methylation and mRNA arrays from xenografts of SKOV3 tumors. Based on both analyses, we demonstrated that BARD1 was one of the top candidate which was highly expressed (p=0.02) and less methylated (p<0.05) in the combination of B20 and 5`-Aza group compared to B20 alone. Our data also showed that BARD1 expression was restored in the combination therapy. The sequential treatment with 5`-Aza reverted the resistance towards the AVA therapy in SKOV3 model. Furthermore, the combination of B20 and 5`-Aza significantly decreased tumor weight in SKOV3 (p=0.005), IG10 (p<0.05), MDA-OVCA1 (p=0.01), and HT1080 (p<0.05). BARD1 expression levels were substantially increased in combination therapy compared to the B20 alone group (>4-fold, p=0.006). Tumor weight and BARD1 expression were inversely correlated. To determine the biological significance of BARD1, we knockdown BARD1 using Tet-inducible shRNA system. Our data showed a significant reduction in tumor weight in the BARD1 knockdown group in SKOV3 model (p=0.021). RNA-sequencing of BARD1 knockdown showed an enrichment of the angiogenesis pathway. Moreover, BARD1 expression levels were decreased in hypoxic conditions both in vitro and in vivo. Conclusion: Our findings indicate a previously unrecognized role for BARD1 in mediating resistance to AVA therapy. Use of epigenetic drugs to control the expression of tumor suppressor genes may enhance the efficacy of AVA therapy in OC. Citation Format: Emine Bayraktar, Cristian Rodriguez-Aguayo, Sudhir Kumar, Elaine Stur, Lingegowda S. Mangala, Stephen Baylin, Gabriel Lopez-Berestein, Sunila Pradeep, Anil K. Sood. Epigenetic regulation of BARD1 confers resistance to anti-VEGF therapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6001.

Abstract 4925: Cannabidiol and Cannabis Sativa as a potential treatment in vitro prostate cancer cells silenced with RBBp6 and PC3 xenograft

Abstract Background: Prostate cancer is the second most frequently occurring carcinoma in males worldwide and one of the leading causes of death in men around the world. Recent studies estimate that over 1.4 million males are diagnosed with prostate cancer on an annual basis, with approximately 375 000 succumbing to the disease annually. With current treatments continuing to show severe side effects, there is a need for new treatments. In this study we looked at the effect of cannabis sativa extract, cannabidiol and cisplatin on prostate cancer cells, PC3. Methods: In addressing the above questions, we employed the MTT assay to measure the antiproliferative effect on PC3 cells following treatment with varying concentrations of Cannabis sativa extract, cisplatin and cannabidiol. xCELLigence was also used to confirm the IC50 activity in which cells were grown in a 16 well plate coated with gold and monitor cell attachment. Caspase 3/7 activity was also measured using 96 well-plate following treatment. Western-blot and qRT-PCR was also used to measure the gene expression of tumor suppressor genes, p53, Bax and Bcl2. Animal studies were employed to measure the growth of PC3-mouse derived cancer to evaluate the effect of compounds in vivo. Results: From the treatment with varying concentrations of Cannabis sativa extract, cannabidiol and cisplatin, we have observed that the three compounds induced antiproliferation of PC3 cancer cell lines through the activation of caspase 3/7 activity. We also observed induction of apoptosis in these cells following silencing of retinoblastoma binding protein 6 (RBBP6), with upregulation of p53 and bax mRNA expression, and a reduction in Bcl2 gene expression. The growth of tumors in the mouse models were reduced following treatment with cisplatin and cannabidiol. Conclusion: We demonstrated that cannabidiol is a viable therapy to treat prostate cancer cells, in combination with silencing of RBBP6. This suggests that cannabidiol rather Cannabis sativa extract may play an important role in reducing cancer progression. Citation Format: Lesetja Raymond Motadi, Zodwa E. Jantjies. Cannabidiol and Cannabis Sativa as a potential treatment in vitro prostate cancer cells silenced with RBBp6 and PC3 xenograft. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4925.

Abstract 828: DNA origami nanostructures as a targeted payload delivery system

Abstract DNA origami is a platform nanotechnology allows for the generation of nanostructures with precisely defined geometric shape that may be easily functionalized with a variety of payloads for therapeutic delivery. These payloads include nucleic acids (miRNA, siRNA, gene sequences), associated Cas protein molecules, therapeutic small molecules drugs, functional peptides (adjuvants or cell penetrating peptides), and incorporated targeting and/or therapeutic antibodies. Hence, DNA origami is a highly promising vehicle to deliver targeted therapeutic payloads to cancer cells or other diseases. Previous studies by our laboratory and others revealed DNA origami mediated drug delivery efficiently delivered daunorubicin or doxorubicin to cancer cells while outperforming free drug in both solid and liquid tumor model systems in vitro and in vivo. Our recent findings showed significant survival advantage by doxorubicin-loaded DNA origami over delivery of free doxorubicin in an aggressive, acute monocytic leukemia model. We also showed antibody-directed drug delivery by DNA origami may be directly targeted to monocytic leukemia cells expressing the target surface antigen, and superior efficacy in vitro against acute myeloid leukemia cells when loaded with daunorubicin relative to free drug at low concentrations. Thus, antibody targeted drug loaded DNA origami represent a promising novel precision medicine approach. Additionally, recent findings by our laboratory in vivo determined that DNA origami nanostructures alone administered at high dose (12.0mg/kg) distribute well, are non-toxic, and illicit mild immunogenicity, making them an attractive candidate for further development towards therapeutic applications. We recently developed a DNA origami structure functionalized at a high density with adjuvant (CpG) and antigenic peptides (OVA) to stimulate a directed antigen-specific immune response. We have shown this DNA origami vaccine platform displays significant improvement in efficacy by an OVA-pulsed antigen-killing assay in vivo relative to alum and CFA standard adjuvant formulations. Thus, our DNA origami vaccine platform represents a promising novel approach to optimize an anti-cancer immune response, a finding also shown by other laboratories.Collectively, DNA Nanobot technology offers 1) a promising a cancer drug delivery system that can deliver a high number of therapeutic drugs (100s of drug molecules per targeted delivery system) utilizing existing ADC conjugation methods; 2) An optimal vaccine delivery method to enhance efficacy; and 3) cell specific targeted delivery of therapeutic nucleic acids and genes to modify oncogene and/or tumor suppressor gene expression. Citation Format: Patrick Halley, Niksa Roki, Nicholas Vantangoli, Tom Zupancic, Jeff Spitzner, Meixiao Long, Karilyn Karilyn Larkin, John Byrd, Carlos Castro, Chris R. Lucas. DNA origami nanostructures as a targeted payload delivery system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 828.

Abstract 6267: A novel and potent EZH1/2 dual inhibitor, HM97662 demonstrates antitumor activity in T-cell lymphoma

Abstract Chromatin remodeling is a crucial process for transcriptional regulation, of which dysregulation is often observed in various human cancers. The enhancer of zeste homology 2 (EZH2) and its homolog EZH1 are catalytic subunits of polycomb repressive complex 2 (PRC2), which trimethylate histone H3 at lysine 27 (H3K27me3) to repress transcription of its target genes. Although methlytransferase activity of PRC2 is mainly contributed by EZH2, EZH1 also conducts a compensatory role to maintain tri-methylation of H3K27. EZH1 also directly binds to chromatin and modulates its condensation. Recent studies have suggested that EZH1 as well as EZH2 played a critical role in T-cell lymphoma such as ATL/L and PTCL, which had high innate EZH1 and increased EZH2 expression upon acquisition of their malignancy. Consequently, dual inhibition of EZH1/2 might induce higher expression of downstream tumor suppressor genes than blocking EZH2 alone, expecting greater activity as an anti-cancer therapy. Herein, we presented a novel and potent EZH1/2 dual inhibitor, HM97662, which simultaneously inhibited the methyltransferase activity of both EZH1 and EZH2 with 2.1 and 16 nM of IC50, respectively. Surface plasmon resonance (SPR) assay verified that HM97662 had great binding affinity on EZH1-EED-SUZ12 complex as well as EZH2-EED-SUZ12 complex than competitors. HM97662 also showed broad and strong antiproliferative activity against various T-cell lymphoma cell lines. Representatively in HH cells, HM97662 not only suppressed global tri-methylation of H3K27, but also dose-dependently increased protein expression levels that modulate cell cycle or cancer cell apoptosis. HM97662 exhibited increment of CDKN1A (p21) proteins involved in cell cycle arrest and induced protein levels of cleaved caspase-3 as well as PARP. Additionally, resulting induction of apoptosis by HM97662 in HH cells was confirmed by TUNEL staining. Mechanistically, HM97662 increased mRNA expression of several target genes inducing cell cycle arrest and apoptosis in gene panel assay performed in HH cells. Two cell cycle repressor, CDKN2A (p16) and CDKN1C (p57), and a pro-apoptotic marker, BTG-2, were dose-dependently increased by HM97662. We further conducted chromatin accessibility assay and identified that the chromatin structures of them were loosened and highly transcribed after the treatment of HM97662. Based our in vitro pharmacology data, we evaluated an antitumor activity of HM97662 in EZH1/2 co-expressed HuT-102 T-cell lymphoma cell mouse xenograft model, and daily oral dosing of HM97662 showed potent tumor growth inhibition. In conclusion, the present studies demonstrated that HM97662 has promising prospective for the treatment of patients with T-cell lymphoma. Given that the necessity of new treatment options on T-cell lymphoma, it is urgent to assess the effectiveness of HM97662 in further clinical trials. Citation Format: Jooyun Byun, Seung Hyun Jung, Yu-Yon Kim, Miyoung Lee, Gunwoo Lee, Heesun Moon, Eun Young Lee, Junghwa Park, Seon Yeong Han, Young Gil Ahn, Young Hoon Kim, Kwee Hyun Suh. A novel and potent EZH1/2 dual inhibitor, HM97662 demonstrates antitumor activity in T-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6267.