Expression Of CDK4 Research Articles

Effects of polyphyllin Ⅶ on proliferation, apoptosis and cell cycle of diffuse large B-cell lymphoma cells

The aim of this study was to investigate the effects of polyphyllin Ⅶ (PP Ⅶ) on proliferation, apoptosis, and cell cycle of diffuse large B-cell lymphoma (PLBCL) cell lines U2932 and SUDHL-4. The DLBCL cell lines were divided into a control group and a PPⅦ group, and experiments were conducted using MTT assay, flow cytometry, and Western blotting.Results showed that compared with the control group, PPⅦ significantly inhibited the proliferation of U2932 and SUDHL-4 cells (P<0.05). Apoptosis assays demonstrated that treatment with 0.50 and 1.00 µmol/L PP Ⅶ significantly increased the apoptosis rates of both cell lines (P<0.05), upregulated apoptosis-related proteins, and downregulated Bcl-2 protein level (P<0.05). Cell cycle analysis revealed that PPⅦ treatment led to an increase in G0/G1-phase cells (P<0.05) and a decrease in G2/M-phase cells (P<0.05), significantly downregulated cyclin D1, CDK4, CDK6, and survivin protein expression (P<0.05). In conclusion, PPⅦ exerted anti-lymphoma effects by inhibiting proliferation, promoting apoptosis, and inducing G0/G1 phase arrest in DLBCL cells.

HOXA9 gene inhibits proliferation and differentiation and promotes apoptosis of bovine preadipocytes

BackgroundHox gene family is an important transcription factor that regulates cell process, and plays a role in the process of adipocytes differentiation and fat deposition. Previous transcriptome sequencing studies have indicated that the Homeobox A9 gene (HOXA9) is a candidate gene for regulating the process of bovine lipid metabolism, but the function and specific mechanism of action remain unclear. Therefore, this study aims to explore the role of HOXA9 in the proliferation, differentiation and apoptosis of bovine preadipocytes through gain-of-function and lose-of-function.ResultIt found HOXA9 highly expressed in bovine adipose tissue, and its expression level changed significantly during adipocytes differentiation process. It gave a hint that HOXA9 may be involved in the process of bovine lipid metabolism. The results of HOXA9 gain-of-function experiments indicated that HOXA9 appeared to act as a negative regulator not only in the differentiation but also in the proliferation of bovine preadipocytes, which is mainly reflected that overexpression of HOXA9 down-regulate the mRNA and protein expression level of PPARγ, CEBPα and FABP4 (P < 0.05). The mRNA expression level of CDK1, CDK2, PCNA, CCNA2, CCNB1, CCND1 and CCNE2, as well as the protein expression of CDK2 also significantly decreased. The decrease of lipid droplets content was the main characteristic of the phenotype (P < 0.01), which further supported the evidence that HOXA9 was a negative regulator of preadipocytes differentiation. The decrease of cell proliferation rate and EdU positive rate, as well as the limitation of transition of preadipocytes from G0/G1 phase to S phase also provided evidence for the inhibition of proliferation. Apart from this above, we noted an interesting phenomenon that overexpression of HOXA9 showed in a significant upregulation of both mRNA and protein level of apoptosis markers, accompanied by a significant increase in cell apoptosis rate. These data led us not to refute the fact that HOXA9 played an active regulatory role in apoptosis. HOXA9 loss-of-function experiments, however, yielded the opposite results. Considering that HOXA9 acts as a transcription factor, we predicted its target genes. Dual luciferase reporter assay system indicated that overexpression of HOXA9 inhibits activity of PCNA promoter.ConclusionTaken together, we demonstrated for the first time that HOXA9 played a role as a negative regulatory factor in the differentiation and proliferation of preadipocytes, but played a positive regulatory role in apoptosis, and it may play a regulatory role by targeting PCNA. This study provides basic data for further exploring the regulatory network of intramuscular fat deposition in bovine.

The Clinicopathological Significance of the Cyclin D1/E1-Cyclin-Dependent Kinase (CDK2/4/6)-Retinoblastoma (RB1/pRB1) Pathway in Epithelial Ovarian Cancers.

Cyclin-dependent kinases (CDK2, CDK4, CDK6), cyclin D1, cyclin E1 and phosphorylated retinoblastoma (pRB1) are key regulators of the G1/S cell cycle checkpoint and may influence platinum response in ovarian cancers. CDK2/4/6 inhibitors are emerging targets in ovarian cancer therapeutics. In the current study, we evaluated the prognostic and predictive significance of the CDK2/4/6-cyclin D1/E1-pRB1 axis in clinical ovarian cancers (OC). The CDK2/4/6, cyclin D1/E1 and RB1/pRB1 protein expression were investigated in 300 ovarian cancers and correlated with clinicopathological parameters and patient outcomes. CDK2/4/6, cyclin D1/E1 and RB1 mRNA expression were evaluated in the publicly available ovarian TCGA dataset. We observed nuclear and cytoplasmic staining for CDK2/4/6, cyclins D1/E1 and RB1/pRB1 in OCs with varying percentages. Increased nuclear CDK2 and nuclear cyclin E1 expression was linked with poor progression-free survival (PFS) and a shorter overall survival (OS). Nuclear CDK6 was associated with poor OS. The cytoplasmic expression of CDK4, cyclin D1 and cyclin E1 also has predictive and/or prognostic significance in OCs. In the multivariate analysis, nuclear cyclin E1 was an independent predictor of poor PFS. Tumours with high nuclear cyclin E1/high nuclear CDK2 have a worse PFS and OS. Detailed bioinformatics in the TCGA cohort showed a positive correlation between cyclin E1 and CDK2. We also showed that cyclin-E1-overexpressing tumours are enriched for genes involved in insulin signalling and release. Our data not only identified the prognostic/predictive significance of these key cell cycle regulators but also demonstrate the importance of sub-cellular localisation. CDK2 targeting in cyclin-E1-amplified OCs could be a rational approach.

Therapeutic Effects of Arctiin on Alzheimer's Disease-like Model in Rats by Reducing Oxidative Stress, Inflammasomes and Fibrosis.

Alzheimer's disease (AD) affects approximately 50 million people globally and is expected to triple by 2050. Arctiin is a lignan found in the Arctium lappa L. plant. Arctiin possesses anti-proliferative, antioxidative and anti-adipogenic. We aimed to explore the potential therapeutic effects of Arctiin on rats with AD by evaluating the expression of TLR4, NLRP3, STAT3, TGF-β, cyclin D1, and CDK2. AD was induced in rats by administering 70 mg/kg of aluminum chloride through intraperitoneal injection daily for six weeks. After inducing AD, some rats were treated with 25 mg/kg of Arctiin daily for three weeks through oral gavage. Furthermore, to examine the brain tissue structure, hippocampal sections were stained with hematoxylin/eosin and anti-TLR4 antibodies. The collected samples were analyzed for gene expression and protein levels of TLR4, NLRP3, STAT3, TGF-β, cyclin D1, and CDK2. In behavioral tests, rats showed a significant improvement in their behavior when treated with Arctiin. Microimages stained with hematoxylin/eosin showed that Arctiin helped to improve the structure and cohesion of the hippocampus, which was previously impaired by AD. Furthermore, Arctiin reduced the expression of TLR4, NLRP3, STAT3, TGF-β, cyclin D1, and CDK2. Arctiin can enhance rats' behavior and structure of the hippocampus in AD rats. This is achieved through its ability to reduce the expression of both TLR4 and NLRP3, hence inhibiting the inflammasome pathway. Furthermore, Arctiin can improve tissue fibrosis by regulating STAT3 and TGF-β. Lastly, it can block the cell cycle proteins cyclin D1 and CDK2.

Inhibition of Proliferation and Induction of Apoptosis in Prostatic Carcinoma DU145 Cells by Polysaccharides from Yunnan Rosa roxburghii Tratt.

This study aimed to investigate methodologies for the extraction and purification of polysaccharides from Rosa roxburghii Tratt fruits and their impact on various cellular processes in prostate cancer DU145 cells, including survival rate, migration, invasion, cell cycle, and apoptosis. Compared to the control group, the polysaccharide exhibited a significant reduction in the viability, migration, and invasion rates of DU145 cells in a time- and dose-dependent manner within the polysaccharide-treated groups. Additionally, it effectively arrested the cell cycle of DU145 cells at the G0/G1 phase by downregulating the expressions of CDK-4, CDK-6, and Cyclin D1. Furthermore, it induced apoptosis by upregulating the expressions of Caspase 3, Caspase 8, Caspase 9, and BAX. Polysaccharides were extracted from Rosa roxburghii Tratt sourced from Yunnan, China. Extraction and decolorization methods were optimized using response surface methodology, based on a single-factor experiment. Polysaccharide purification was carried out using DEAE-52 cellulose and Sephadex G-100 column chromatography. The optimal dosage of R. roxburghii Tratt polysaccharide affecting DU145 cells was determined using the CCK-8 assay. Cell migration and invasion were assessed using transwell and scratch assays. Flow cytometry was employed to analyze the effects on the cell cycle and apoptosis. Western blotting and Quantitative real-time PCR were utilized to examine protein and mRNA expressions in DU145 cells, respectively. Rosa roxburghii Tratt polysaccharides, consisting of D-mannose, L-rhamnose, N-acetyl-D-glucosamine, D-galacturonic acid, D-glucose, D-galactcose, D-xylose, L-arabinose, and L-fucose, possess the ability to hinder DU145 cell proliferation, migration, and invasion while inducing apoptosis through the modulation of relevant protein and gene expressions.

LncRNA BACE1-AS Accelerates the Progression of Gastric Cancer Through Regulating as a ceRNA of miR-422a to Positively Control BRD4 Expression

Gastric cancer (GC) is a leading global cause of cancer-related mortality, necessitating urgent research on its pathogenesis, prevention, and treatment. In this study, we investigated the expressions of LncRNA BACE1-AS, mRNA BRD4, and miR-422a in GES-1 and GC cells under various treatments using RT-PCR. Western Blots confirmed protein expressions in HGC-27 and SNU-1 cells. EDU and MTT assays assessed cell proliferation, while Transwell tests determined invasion capacity, and flow cytometry analyzed apoptosis. BACE1-AS and BRD4 were significantly elevated in cancerous tissues compared to paired non-cancerous tissues. BACE1-AS knockdown inhibited invasion and proliferation, promoting apoptosis. miR-422a mimics suppressed proliferation and invasion while enhancing apoptosis, and miR-422a mimics with BRD4 overexpression had the opposite effect. Moreover, BAX protein increased in the si-BACE1-AS group but decreased in the si-BACE1-AS+miR-422a inhibitor group. Si-BACE1-AS and miR-422a mimics reduced the expression of C-Myc, CyclinD1, Survivin, CDK4, and Bcl-2, while the si-BACE1-AS+miR-422a inhibitor and miR-422a mimics+BRD4-OV groups showed the opposite trend. Our findings suggest that LncRNA BACE1-AS positively regulates gastric cancer progression by modulating BRD4 as a competitive endogenous RNA for miR-422a. This LncRNA BACE1-AS/BRD4/miR-422a signaling axis presents potential targets for developing therapeutic strategies against gastric cancer.

Gonadal transcriptome analysis of genes related to sex differentiation and sex development in the Pomacea canaliculata

As an invasive alien animal, Pomacea canaliculata poses a great danger to the ecology and human beings. Recently, there has been a gradual shift towards bio-friendly control. Based on the development of RNA interference and CRISPR technology as molecular regulatory techniques for pest control, it was determined if the knockout of genes related to sex differentiation in P. canaliculata could induce sterility, thereby helping in population control. However, the knowledge of sex differentiation- and development-related genes in P. canaliculata is currently lacking. Here, transcriptomic approaches were used to study the genes expressed in the two genders of P. canaliculata at various developmental stages. Gonad transcriptomes of immature or mature males and females were compared, revealing 12,063 genes with sex-specific expression, of which 6066 were male- and 5997 were female-specific. Among the latter, 581 and 235 genes were up-regulated in immature and mature females, respectively. The sex-specific expressed genes identified included GnRHR2 and TSSK3 in males and ZAR1 and WNT4 in females. Of the genes, six were involved in reproduction: CCNBLIP1, MND1, DMC1, DLC1, MRE11, and E(sev)2B. Compared to immature snail gonads, the expression of HSP90 and CDK1 was markedly reduced in gonadal. It was hypothesized that the two were associated with the development of females. These findings provided new insights into crucial genetic information on sex differentiation and development in P. canaliculata. Additionally, some candidate genes were explored, which can contribute to future studies on controlling P. canaliculata using molecular regulatory techniques.

Anticancer potential of grifolin in lung cancer treatment through PI3K/AKT pathway inhibition

ObjectiveGrifolin is a natural secondary metabolite isolated from edible fruiting bodies of the mushroom Albatrellus confluens. Grifolin has antitumor activities in several types of cancer. We aimed to determine the effects of grifolin on lung cancer. MethodsWe determined the proliferation, migration, invasion, and apoptosis of lung cancer cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Ethynyl deoxyuridine, colony formation, wound scratch, transwell, flow cytometry, and xenograft mouse assays. Molecular docking evaluated the binding relation between grifolin and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA). The levels of PIK3CA, AKT, and p-AKT were measured by western blot. ResultsGrifolin (10, 20, or 40 μM) inhibited the proliferation, migration, and invasion of lung cancer cells, and induced cell cycle arrest and apoptosis. Grifolin also decreased CDK4, CDK6, and CyclinD1 expression and significantly decreased PIK3CA and p-AKT expression in lung cancer cells. These anticancer effects were abolished by 740Y-P. ConclusionsGrifolin regulates the PI3K/AKT pathway, thus inhibiting lung cancer progression.

CircDDX17 targets miR-223-3p / RIP3 to regulate the proliferation and apoptosis of non-small cell lung cancer cells

Objective: To explore the molecular mechanism of circDDX17 regulating the proliferation and apoptosis of non-small cell lung cancer cells by targeting the miR-223-3p/RIP3 molecular axis. Methods: The expression levels of circDDX17, miR-223-3p, and RIP3 in human normal lung epithelial cell lines BEAS-2B and non-small cell lung cancer cells H1299, A549, and H446 were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The plasmids of pcDNA, pcDNA-circDDX17, anti-miR-con, anti-miR-223-3p, pcDNA-circDDX17 and miR-con, pcDNA-circDDX17 and miR-223-3p mimics were transfected into H1299 cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay was used to detect the cell proliferation. Flow cytometry was used to detect the cell cycle and cell apoptosis. Plate cloning experiment was used to detect cell proliferation ability. The dual luciferase report experiment was applied to verify the targeting relationship between miR-223-3p with circDDX17 and RIP3. Western blot was used to detect the protein expression of cyclinD1, CDK2, cleaved caspase-3 and Bax. Results: The expression levels of circDDX17 and RIP3 mRNA in H1299, A549, and H446 cells were significantly reduced (P<0.05), the expression level of miR-223-3p mRNA was significantly increased (P<0.05) compared with BEAS-2B. The cell viability [(69.46±4.68)%], the number of cell clones (83.49±7.86), the proportion of cells in S phase [(22.52±1.41) %], the protein expression levels of cyclinD1 and CDK2 in PCDNa-CircDDX17 group were lower than those in pcDNA group [(97.54±7.72)%, 205.03±13.37, (28.69±1.49)%, respectively, P<0.05], while the percentage of G0/G1 phase cells [(64.45±3.56)%], apoptosis rate [(18.36±1.63)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17 group were higher than those of pcDNA group [(51.33±2.76) % and (5.21±0.54) %, respectively, P<0.05]. The viability [(72.64±5.44)%], the number of cell clones (78.16±8.23), the proportion of S-stage cells [(21.34±1.59) %], the protein expression levels of CyclinD1 and CDK2 in anti-miR-223-3p group were lower than those in anti-miR-con group [(103.47±6.25)%, 169.32±14.53, (28.43±1.26)%, respectively, P<0.05]. Percentage of G0/G1 phase cells [(62.86±3.28)%], apoptosis rate [(14.64±1.67)%], the protein expression levels of cleaved caspase-3 and Bax in the anti-miR-223-3p group were higher than those of anti-miR-con group [(51.33±2.71)% and (4.83±0.39)%, respectively, P<0.05]. MiR-223-3p has complementary sites with circDDX17 or RIP3. The viability [(135.45±9.28)%], the number of cell clones (174.64±10.68), the proportion of S-phase cells [(26.39±2.25)%], the protein expression levels of cyclinD1 and CDK2 in pcDNA-circDDX17+miR-223-3p group were higher than those in pcDNA-circDDX17+miR-con group [(101.56±6.68)%, 107.65±7.62, (21.64±1.72)%, P<0.05]. Percentage of G0/G1 phase cells [(56.64±2.76)%], apoptosis rate [(8.34±0.76)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17+miR-223-3p group were lower than those of pcDNA-circDDX17+miR-con group [(64.03±3.48)% and (15.21±1.18)%, respectively, P<0.05]. Conclusion: circDDX17 could inhibit the proliferation and induce apoptosis of non-small cell lung cancer cells via targeting the miR-223-3p / RIP3 molecular axis.

Abstract 249: Deciphering the impact of TRIB3-ELF4 interaction in regulating CDK6 on cancer stem cell activity in endometrial cancer

Abstract Background: Endometrial cancer (EC) is the most common gynecological tumor among women worldwide, posing a serious threat to women's health. ELF4 (E74 like ETS transcription factor 4), a member of the ETS family, plays a role in facilitating cancer progression. Our previous work demonstrated the role of direct binding between ELF4 and tribbles pseudokinase 3 (TRIB3) in promoting β-catenin expression. In this study, we further explore ELF4's involvement in the maintenance of cancer stem cells (CSCs) in EC. Methods: The vital role of ELF4 in EC was determined through gain- and loss-of-function assays. ELF4 was silenced in two EC cell lines, AN3CA and HEC-1A, and primary EC cells from a Taiwanese patient (EMC6), using RNA interference techniques. The expression of ELF4, TRIB3, and CDK6 in human EC samples was assessed using immunohistochemistry. Tumorsphere formation assays, quantitative real-time PCR, and immunoblotting quantified CSC activity and the involvement of related signaling pathways. Transcriptional regulation associated with ELF4 was investigated using luciferase reporter assays and chromatin immunoprecipitation. Results: Analysis of the TCGA database via the TISIDB web tool revealed that elevated ELF4 levels correlated with higher histological grades, advanced clinical stages, and poorer overall survival in EC patients. A grade-dependent increase in ELF4 expression was observed using tissue microarrays. Knockdown of ELF4 in EC cell lines and patient-derived EC cells significantly reduced cell proliferation, cell cycle progression, migration, invasion, and CSC activity. RNA-seq data analysis of ELF4-knockdown EC cells identified CDK6 as a potential downstream target of the ELF4/TRIB3 interaction. Knockdown of ELF4 and TRIB3 significantly downregulated CDK6 expression. Co-expression of TRIB3 and ELF4 enhanced CDK6 expression, and their expression was positively associated with CDK6 in EC specimens. We also demonstrated that CDK6 facilitates EC-CSC activity. Knockdown of CDK6 significantly attenuated tumorsphere formation and reduced the levels of cancer stemness-related proteins, including OCT4, NANOG, and c-MYC. Conclusion: Our study demonstrates the essential roles of ELF4 and TRIB3 in regulating CDK6 expression, contributing to the maintenance of EC-CSCs. Citation Format: Wen-Ling Wang, Wen-Wei Chang. Deciphering the impact of TRIB3-ELF4 interaction in regulating CDK6 on cancer stem cell activity in endometrial cancer [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 249.

Abstract 2493: Prospective evaluation of predictive biomarkers for Palbociclib (P) and endocrine therapy (ET) benefit in hormone receptor (HR)-positive [+]/human epidermal growth factor receptor 2 (HER2)-negative [-] advanced breast cancer (ABC) patients (pts) from PARSIFAL clinical trial: The transFAL sub-study

Abstract BACKGROUND Combining cyclin-dependent kinases 4 and 6 inhibitors (CDK4/6i) with first-line ET is the standard of care for pts with HR[+]/HER2[-] ABC. PARSIFAL was a randomized, open-label, phase II trial that evaluated the efficacy and safety of first line P plus fulvestrant (F) or letrozole (L) in this pts population. Both treatments had comparable efficacy and safety results (JAMAOncol. 2021;7(12):1791-1799). Although multiple CDK4/6i clinical outcomes biomarkers have been reported, the only validated predictive factor for response is HR positivity. TransFAL, a translational study of PARSIFAL, assessed a comprehensive set of biomarkers of sensitivity/resistance to P plus ET. MATERIALS AND METHODS TransFAL employed a sample biorepository from PARSIFAL comprising formalin-fixed paraffin-embedded (FFPE) tissue and blood samples obtained from pts included in either study arm. Tumor samples (N=33) derived from the primary tumor at initial diagnosis or from primary tumor or metastatic lesions at study entry. Blood samples were obtained at baseline (N=73) and disease progression (N=22). Pts were categorized as sensitive or resistant based on progression-free survival (PFS). CDK4/6 pathway activation was assessed by immunohistochemistry for CDK4, CDK6, Rb, pRb, ER, PgR, and Ki67. Proteins extracted from FFPE slides were analyzed using hierarchical clustering, significance analysis of microarrays, and probabilistic graphical models. RNA expression and circulating tumor DNA (ctDNA) analysis were assessed by next-generation sequencing (NGS) and analysis of panel of 56 genes/gene regions, respectively. RESULTS A worse median PFS was found in pts with CDK6 positive (≥ 1% positive cells; p &amp;lt; 0.001) and high Ki67 (≥ 10% positive cells; p = 0.013)at baseline. ctDNA density at baseline was significantly higher for resistant (PFS &amp;lt; 9 months; 17.1 ng/mL) pts compared to sensitive (PFS &amp;gt; 31 months; 11.3 ng/mL) pts (p &amp;lt; 0.001). Most pts (83%) presented a genomic alteration detected in ctDNA, TP53 (28%) and PIK3CA (28%) being the most frequently mutated genes. Pts (9%) who harbored ARID1A mutation had lower PFS (p = 0.052). ESR1 mutations at progression were numerically higher in the L arm (37.5%) compared to F arm (14.3%); (p=0.31), but numbers were low. Proteomic and RNA analyses did not yield confirmable distinctive expression pattern between resistant/sensitive pts… CONCLUSIONS High Ki67 levels and CDK6 expression in the tumor and high ctDNA density, but not proteomic or RNA sequencing data, were associated to poor outcome in this analysis of pts treated with CDK4/6i and ET. This data adds to the growing evidence of poor prognosis biomarkers in this setting, highlighting the need of novel strategies for these pts. Citation Format: Joan Albanell, Angelo Gámez Pozo, Carlos L. Arteaga, Meritxell Bellet, Federico Rojo, Abel González, Beatriz Bellosillo, Violeta Serra, Petra Gener, José Antonio Guerrero, Laura López-Montero, Mario Mancino, Jose Rodríguez-Morató, Leonardo Mina, José Manuel Pérez-García, Javier Cortés, Antonio Llombart-Cussac. Prospective evaluation of predictive biomarkers for Palbociclib (P) and endocrine therapy (ET) benefit in hormone receptor (HR)-positive [+]/human epidermal growth factor receptor 2 (HER2)-negative [-] advanced breast cancer (ABC) patients (pts) from PARSIFAL clinical trial: The transFAL sub-study [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 2493.

Abstract 3044: Novel agents with efficacy against germline mutant RUNX1 familial platelet disorder with myeloid malignancy (FPD-MM)

Abstract RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Germline, mono-allelic, missense mutations in RUNX1 cause large deletions or truncations that are mostly loss-of-function mutations. They cause familial platelet disorder (RUNX1-FPD), which evolves into myeloid malignancy (FPD-MM), e.g., MDS or AML. Although curative in some patients, allogeneic stem cell transplantation from matched, un-related donors carries the risk of GVHD and relapse in FPD-MM. Thus, there is a need for novel therapies to revert FPD-MM back to FPD. LINCS1000-CMap analysis, of the RUNX1 knockdown RNA-Seq signature revealed homoharringtonine (HHT or omacetaxine) and the anthelmintic fenbendazole (analog of mebendazole (MB)) as the top expression mimickers. In our current studies, we demonstrate that treatment with HHT or MB induced significantly greater loss of viability in seven patient-derived samples of FPD-MM vs RUNX1-FPD. Analyses conducted on BMA cells harvested longitudinally from the same patient at the RUNX1-FPD versus FPD-MM stage demonstrated greater chromatin accessibility in FPD-MM cells along with increased mRNA expressions of MYB, MECOM and BCL2. Following HHT treatment, scRNA-Seq analysis demonstrated a marked reduction in the HSC population, but concomitant increase in the macrophage population in FPD-MM. This was associated with positive enrichment of apoptosis signaling and TP53 targets gene-sets, while negatively enriching those of c-Myc targets. CyTOF analysis demonstrated that HHT treatment reduced c-Myc, EVI1, MCL1, BFL1 and CDK6 expression in phenotypically characterized FPD-MM stem-progenitor cells. Utilizing an FPD-MM sample harboring Runx1 K194N, also detected in other pedigree members with FPD or FPD-MM, we established the first ever cell line (GMR-AML1) expressing germline mtRUNX1. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL-3R) and CD33. GMR-AML1 cells in which Runx1 was knocked-out by CRISPR-gRNA were outcompeted in vitro and in vivo, by cells that retained Runx1. Treatment with HHT or MB induced loss of viability in GMR-AML1 cells (LD50: 40 or 330 nM, respectively). HHT also caused concordant decline in ATAC-Seq and RNA-Seq peaks in ribosomal genes involved in protein translation. Treatment with MB inhibited cell cycle and protein translation. CyTOF analysis revealed that MB treatment also depleted CDK6, RUNX1, c-Myc and EVI1 in phenotypically defined FPD-MM stem cells. Finally, in the GMR-AML1 cell xenograft, treatment with omacetaxine or MB significantly reduced AML burden and improved overall survival of NSG mice. These preclinical findings highlight the molecular features associated with progression of RUNX1-FPD to FPD-MM and highlight HHT or MB as effective against cellular models of FPD-MM. Citation Format: Christopher P. Mill, Warren C. Fiskus, Courtney D. DiNardo, Patrick K. Reville, John A. Davis, Christine E. Birdwell, Kaberi Das, Hanxi Hou, Koichi Takahashi, Sanam Loghavi, Joseph D. Khoury, Kapil Bhalla. Novel agents with efficacy against germline mutant RUNX1 familial platelet disorder with myeloid malignancy (FPD-MM) [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 3044.

Abstract 5960: Targeting CDK8: A translatable therapeutic approach for fusion-positive aRMS

Abstract Alveolar rhabdomyosarcoma (aRMS), characterized by poor overall survival and limited advancements in therapy over the past four decades, poses a great challenge in childhood cancer treatment. More than 60% of high-risk aRMS is driven by the oncofusion protein PAX3-FOXO1. The direct targeting of transcription factor fusions, however, is quite difficult. Given that PAX3-FOXO1 is reported to drive disease by controlling transcriptional programs to maintain an undifferentiated myoblastic state, an alternative approach would be to target the transcriptional machinery recruited by the fusion. To identify potential novel targets in aRMS, we utilized the Broad Institute’s Cancer Dependency Map dataset, a collection of genome-wide CRISPR-Cas9 screens performed in over 1000 cancer cell line models. We focused on druggable dependencies that are selective to fusion-positive aRMS and intersected this data with PAX3-FOXO1-associated transcriptional complexes. Among these candidates, we identified CDK8 as a top selective dependency. Importantly, other components of the CDK8 kinase module in the mediator complex, such as CCNC, also scored as a strong selective dependency in fusion-positive aRMS. CDK8 expression is notably elevated in aRMS tumors compared to other pediatric tumors, and aRMS cell lines exhibit significantly higher CDK8 protein levels than non-transformed primary skeletal muscle cells. We next validated these in silico results through the application of shRNA knockdown, CRISPR knockout, and the use of CDK8 kinase inhibitory small molecules, including BI-1347, SEL-120-34A, and JH-XII-178, which demonstrated on-target activity as assessed by STAT1Ser727 phosphorylation. Both genetic loss of CDK8 function and kinase inhibition impaired aRMS cell line growth in vitro. In vivo studies demonstrated a significant inhibition of xenograft tumor growth with both doxycycline inducible CDK8-directed shRNAs and with pharmacologic inhibition of CDK8 with SEL120-34A treatment. To understand the mechanisms by which CDK8 inhibition impairs aRMS growth, we evaluated the transcriptional consequences of CDK8 inhibition by RNA-sequencing in aRMS, as well as the chromatin accessibility by ATAC-sequencing. We discovered that loss of CDK8 led to gain of chromatin accessibility and the upregulation of hallmark genes involved in myogenesis, such as MYH3, MYOG, MEF2C, and MYBPH, as well as alteration of PAX3-FOXO1 gene expression programs. We next validated this differentiation phenotype both in vitro through detection of increased myogenin staining by immunofluorescence and in vivo by visualization of myofibrils in the xenograft tumors and increased myoglobin quantified by qRT-PCR after CDK8 inhibition. In conclusion, this work identifies CDK8 as a novel and readily translatable therapeutic target in fusion-positive aRMS laying a robust foundation for further mechanistic and preclinical studies. Citation Format: Susu Zhang, Clare Malone, Brian Guedes, Kathleen Engel, Marissa Just, Kenneth Ross, Assil Fahs, Giulia Digiovanni, Diyana Granum, Rex Bentley, Leen Barbar, Christian Cerda-Smith, Elizabeth Mendes, Ozgun Erdogan, Alexander Kovach, Kristianne Oristian, Seth Zimmerman, Jack F. Shern, Kris Wood, Chris Counter, Corinne M. Linardic, Kimberly Stegmaier. Targeting CDK8: A translatable therapeutic approach for fusion-positive aRMS [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 5960.

Abstract 1227: Novel combination therapies to overcome non-genetic/adaptive menin inhibitor resistance in AML with MLL1r or mtNPM1

Abstract In AML with MLL1 rearrangement or MLL1 fusion proteins (FP), the N-terminus of MLL1 becomes fused to over 80 partner proteins resulting in an aberrant leukemic transcriptional program including dysregulated expression of HOXA9, MEIS1, PBX3, FLT3, MEF2C and CDK6. On the other hand, in mutant NPM1 AML cells, wild type MLL1 is the main regulator of HOXA9, MEIS1 and FLT3, promoting self-renewal of myeloid progenitor cells. Menin inhibitors have been developed that disrupt the binding of Menin to MLL1 leading to reduced activity of HOXA9 and MEIS1 and repression of MLL1 or MLL1-FP target genes. Clinical trials have demonstrated that Menin inhibitors (MI) are well-tolerated and clinical remissions have been observed in patients with relapsed/refractory AML harboring MLLr. Unfortunately, most patients either fail to respond or eventually relapse. MI-resistance in AML with MLL1r or mtNPM1 is either due to recently described mutations in the MI-binding domain of Menin or is non-genetic/adaptive, due to dysregulated epigenome/transcriptome/proteome. In the present studies, through repeated shocks with LD90 dosing of MI, followed by drug washout and recovery, we have generated MLL1r (MV4-11-MITR) and mtNPM1 (OCI-AML3-MITR) AML cell lines that are tolerant/resistant (TR) to MI with LD50 values greater than 1 μM. Whole exome sequencing confirmed that these cells were lacking in new driver mutations. We determined that resistance in the MITR cells was not due to presence of hotspot mutations in Menin. ChIP-Seq analysis of H3K27Ac showed that the activities of super enhancers and core transcriptional regulatory circuitry was altered in both MITR cell types. Non-genetic/adaptive resistance to MI also concordantly reduced genome wide ATAC-Seq and RNA-Seq peaks in the OCI-AML3-MITR cells with significant depletion of MEIS1, IGF2BP2, BCL2, and PBX3, with concomitant induction of leukemia stem cell associated CLEC12A and CD244 expressions. A domain-specific CRISPR screen in MV4-11-MITR cells identified several druggable co-dependencies (e.g. EP300 and MOZ) and co-enrichments (SMARCA4, CREBBP and BRD4) with MI, suggesting them as potential mechanisms of non-genetic/adaptive MI resistance. Consistent with these findings, co-treatment with SMARCA2/SMARCA4 (chromatin remodeler ATPases) inhibitor FHD-286 and the BETi OTX015 or the MI SNDX-50469 exerted synergistic lethality in MV4-11-MITR and OCI-AML3-MITR cells as well as in PD MITR AML cells with MLL1r or mtNPM1. Co-treatment with CBP/p300 inhibitor GNE049 or GNE781 was also synergistic with MI in these cells. In vivo treatment with FHD-286 and OTX015 or SNDX-5613 significantly reduced the AML burden in mice bearing OCI-AML3-MITR xenografts. These findings underscore preclinical activity of epigenetically-targeted agent-based combinations and highlight their promise in overcoming MI resistance in AML with MLL1r or mtNPM1. Citation Format: Warren C. Fiskus, Christopher P. Mill, Jessica Piel, Murphy Hentemann, Christine E. Birdwell, Kaberi Das, John A. Davis, Hanxi Hou, Tapan M. Kadia, Naval Daver, Sanam Loghavi, Branko Cuglievan, Courtney D. DiNArdo, Kapil N. Bhalla. Novel combination therapies to overcome non-genetic/adaptive menin inhibitor resistance in AML with MLL1r or mtNPM1 [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 1227.

Abstract 506: TRIP13 is a promising therapeutic target for bladder cancer

Abstract Bladder cancer (BCa) is the second most common genitourinary cancer. High recurrence rate, progression to MIBC and poor prognosis in metastatic disease are major challenges for this disease management. Therapeutic options for advanced stage BCa are very limited, hence developing agents to treat this disease progression are highly needed. Thyroid receptor-interacting protein 13 (TRIP13), a member of the AAA-ATPase family, regulates various cellular processes such as DNA repair and spindle assembly. TCGA data analysis showed significant overexpression of TRIP13 that correlates with disease stage and mortality. Recent studies have shown that TRIP13 amplification occurs in several types of human cancers, including BCa, and it regulates cell proliferation, tumor growth, and drug resistance. However, the role of TRIP13 in BCa and its potential as a therapeutic target remain elusive. Therefore, in the present study we performed a preclinical investigation of the TRIP13 role in BCa progression, underlying molecular mechanisms, and develop promising therapeutics against BCa. Initially we profiled TRIP13 expression in 10 different bladder cancer cell lines from a panel of human and rodent origin and found elevated protein expression in multiple BCa cell lines. TRIP13 levels were also significantly elevated in the BBN induced rat- and SV40T driven mouse- bladder tumor tissues compared to normal bladder tissues. Pharmacological inhibition of TRIP13 with a potent drug DCZ0415 significantly suppressed cell proliferation (IC50 ~7μM) in J82, NBT-II and MB49 cell lines and induced cell death. DCZ0415 treatment led to cell cycle arrest at G1 phase (P&amp;lt;0.001) in BCa cells. In mechanistic investigations on its effect on cell cycle progression by DCZ0415 treatment decreased the expression of CDK4, CDK6, Cyclin D1, and PCNA protein expression. TRIP13 inhibition also suppressed the migration and invasion potential of BCa cells. Annexin V and γ-H2AX staining suggested that DCZ0415 induced apoptosis in BCa cells and increased DNA damage. These findings were also complemented by an increase in cleaved caspase-3 and PARP proteins. We also observed modulation of cGAS-STING signaling pathway in BCa cells with DCZ0415 treatment, indicating potential for its combination with cGAS-STING stimulators. Importantly, we found that TRIP13 inhibition with DCZ0415 significantly suppressed the growth of 3D BCa models as well BBN-induced rat BCa tumoroids and spheroids. Overall, these results suggest that TRIP13 could be a potential therapeutic target for prevention of bladder cancer and warrants further validation in in vivo models (Funding supported by P30CA225520 and ACS-IRG grant). Citation Format: Surya Pratap Singh, Krishnendu Goswami, Adam S. Asch, Chinthalapally V. Rao, Venkateshwar Madka. TRIP13 is a promising therapeutic target for bladder cancer [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 506.

Abstract 5909: Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer

Abstract Objectives: Given the intricate relationship between inflammation and ovarian cancer (OC) development and progression, anti-inflammatory drugs may have potential in the prevention and treatment of OC. Sulindac exhibits potent anti-inflammatory effects through inhibition of COX1/COX2 pathways and promising anti-tumor activity in pre-clinical models of colorectal and endometrial cancer. Thus, we investigated the efficacy of sulindac as an anti-tumorigenic agent in OC pre-clinical cell and mouse models. Methods: The human OC cell lines, MES and OVCAR5, and the K18-gT121+/-;p53fl/fl;Brca1fl/fl (KpB) mouse model of high-grade serous OC were used. Cellular proliferation was assessed by MTT and colony count assays. Apoptosis was assessed using cleaved caspase 3, 8, and 9 assays. To measure cellular stress, production of reactive oxygen species was measured by DCFH-DA assay, and the JC1 assay was used to assess changes in mitochondrial membrane potential. Cellular adhesion was evaluated by laminin assay, and cellular migration was determined by wound healing assay. Western immunoblotting was used to assess the effects of sulindac on downstream targets related to cellular stress, apoptosis, cellular invasion potential, cell cycle control, and inflammation. The KpB mice were treated with sulindac (7.5 mg/kg, oral gavage, daily) or placebo for four weeks. Results: Sulindac inhibited cellular proliferation in a dose-dependent manner in the MES and OVCAR5 cells, with IC50s of 75.3 and 75.69 μM, respectively. Sulindac (100 μM) reduced colony formation by 81% in the MES cells and by 49% in the OVCAR5 cells (p&amp;lt;0.01). Treatment of MES and OVCAR5 cells with sulindac increased ROS production and decreased mitochondrial membrane potential, in a dose-dependent manner (p&amp;lt;0.01). Sulindac induced activity of cleaved caspase 3, 8, and 9, in MES and OVCAR5 cells (p&amp;lt;0.01). In addition, sulindac (100 μM) effectively decreased cellular adhesion by 31% in MES cells and by 23% in OVCAR5 cells (p&amp;lt;0.01). These results were confirmed by Western immunoblotting demonstrating that sulindac downregulated the anti-apoptotic proteins Bcl-xl and Mcl-1 and upregulated the pro-apoptotic proteins Bax and cellular stress proteins Bip, ATF-4, and PDI, in both cell lines. Sulindac downregulated expression of several epithelial-mesenchymal transition proteins in both cell lines, including beta-catenin, snail, and slug. Sulindac also downregulated expression of cell cycle proteins CDK4, CDK5, and Cyclin D1 and inflammatory protein COX-2 in both cell lines. After 4 wks of treatment, sulindac significantly inhibited tumor growth by 71% (1.89 g vs 0.54 g, p&amp;lt;0.01) in KpB mice. Conclusions: Our results find that sulindac exhibited anti-tumorigenic effects in OC cell lines and the KpB OC mouse model. Given these promising pre-clinical results, further studies on the repurposing of sulindac for the prevention and treatment of OC is warranted. Citation Format: Nikita Sinha, Shuning Chen, Jennifer Haag, Xiaochang Shen, Boer Deng, Ziyi Zhao, Chunxiao Zhou, Victoria Bae-Jump. Sulindac exhibits anti-proliferative effects in pre-clinical models of ovarian cancer [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 5909.

Abstract 6945: Dissecting the interplay of cell identity and extrachromosomal DNA in glioblastoma

Abstract The five-year survival rate of glioblastoma multiforme (GBM) is 5.8%, and its standard of care has not changed since 2005. This stagnation in therapeutic development is due, in part, to its characteristic heterogeneity–multiforme–highlighting a critical need to understand the biological processes underlying this GBM hallmark. Despite the well-documented individual relationships between extrachromosomal DNA (ecDNA), GBM, and heterogeneity, the interplay between the three is not well understood. The Furnari lab has generated a library of cancer avatars, or human induced pluripotent stem cells (hiPSCs) engineered to harbor cancer driving mutations. Single cell RNA sequencing (scRNA-seq) was used to characterize the longitudinal evolution of the first avatars engineered to have TP53−/−;PDGFRAΔ8-9 and PTEN−/−;NF1−/− genotypes. The TP53−/−;PDGFRAΔ8-9 avatar acquired ecDNA, while the PTEN−/−; NF1−/− avatar did not. Re-analysis of the scRNA-seq data identified a subpopulation of cells in the TP53−/−;PDGFRAΔ8-9 avatar that had high expression of CDK4 and PDGFRA, two genes that are commonly co-amplified, found on ecDNA, and drive cellular states. This subpopulation of cells also had highly expressed genes near the CDK4 locus indicating a structural variant and possibly ecDNA. This population diverged transcriptionally from other cells and was associated with the OPC-like cellular state, which has been shown to be associated with high levels of PDGFRA and, at times, co-expression with CDK4. While TP53−/−;PDGFRAΔ8-9 tumors contained cells across all four GBM cellular states, PTEN−/−;NF1−/− tumors had a significantly less diverse composition of cellular states suggesting that ecDNA may facilitate cellular state diversity. With the eventual goal to directly test whether ecDNA enables transcriptional state heterogeneity compared to chromosomally inherited amplifications, CRISPR-Cas9 and CRISPR-C were implemented to engineer EGFRvIII ecDNA in U87. While inverse PCR confirmed the successful generation of ecDNA in U87, ecDNA was lost following long term in vitro cell passaging. Current follow up work aims to identify different intrinsic (oncogene amplified, genetic background, cell type, cell line) and extrinsic (in vitro conditions, orthotopic engraftment) contexts that promote the selection and amplification of engineered ecDNA, as it would be would be valuable to have genotypically identical cancer models with different classes of amplifications (i.e., engineered ecDNA vs. lentiviral overexpression, which is chromosomally inherited). Together, these results highlight the potential to use hiPSC-derived models and single cell methods to investigate gliomagenesis and cell identity. Citation Format: Brett Taylor, Brandon Jones, Daisuke Kawauchi, Yohei Miyake, Raghav Vadla, Nathan Jameson, Shunichiro Miki, Tomoyuki Koga, Bing Ren, Frank Furnari. Dissecting the interplay of cell identity and extrachromosomal DNA in glioblastoma [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 6945.

Abstract 5711: Anti-tumor effects of fadraciclib, CDK2/9 inhibitor, in biliary tract cancer

Abstract Background: Targeting CDKs has emerged as a significant strategy in the development of new drugs. The CDK4/6 inhibitors have proven their efficacy in several tumor types, while the CDK2 and CDK9 inhibitors are currently undergoing clinical trials. In biliary tract cancer (BTC), gene expressions of CDK2 and CDK9 are elevated compared to normal tissue. CDK9, a transcriptional CDK, regulates RNA polymerase II (RNAP II), leading to enhanced transcription of oncogenes, such as MCL1. Aberrant activation of these CDKs is associated with cancer progression and evading apoptosis in BTC. Notably, MCL1 is frequently amplified in intrahepatic cholangiocarcinoma (16-21%). This finding underscores the potential significance of CDK2 and CDK9 as therapeutic targets in BTC. However, in BTC, targeting CDK2 or CDK9 has not yet been studied. In this study, we aimed to test CDK2/9 targeting and develop possible biomarker strategies in BTC. Method: A total 9 BTC cell lines (SNU245, SNU308, SNU478, SNU869, SNU1196, SNU2670, SNU2773, TFK1, and HUCCT1) were used. Fadraciclib (CDK2/9 dual inhibitor), Olaparib (PARP inhibitor), JQ1 (BRD4 inhibitor) was used. MTT assay, Colony formation assay, Annexin-V assay, and Cell cycle analysis were performed for evaluation of anti-cancer effects. The effect of Fadraciclib on homologous recombination (HR)-mediated DNA damage repair was assessed using focus formation assay and DRGFP assay. Combination of Fadraciclib with Olaparib or JQ1 was evaluated in vitro and in vivo. Result: The anti-tumor effect of Fadraciclib monotherapy was relatively higher in MCL1-High BTC cells (SNU869, SNU2773) compared to MCL-Low cells (SNU245, SNU2670). In MCL1-High cells, Fadraciclib inhibited CDK9, leading to downregulation of RNAP II phosphorylation at Serine 2 and MCL1. Fadraciclib also interfered with HR-mediated DNA damage response by inhibiting transcription of HR-related genes. The combination of Fadraciclib and Olaparib exhibited synergistic anti-tumor effects in vitro in MCL1-High cells and in vivo xenograft model. Conversely, in MCL1-Low cells, Fadraciclib increased BRD4 mRNA expression, leading to the restoration of CDK9-RNAP II activity. This reactivation of RNAP II restored cellular transcription homeostasis, thereby reactivating the transcription of oncogenes such as MCL1. Combination of Fadraciclib and JQ1 showed synergistic effects in vitro in MCL1-Low cells and in vivo xenograft model by inhibiting restored CDK9-RNAP II activity. Conclusion: MCL1-High BTC cells are sensitive to CDK2/9 inhibitor and show synergism between CDK2/9 inhibitor and PARP inhibitor. In MCL1-Low BTC, a combination of a CDK2/9 inhibitor and a BRD4 inhibitor might represent an optimal strategy for new drug development. Citation Format: Jae-Min Kim, Ah-Rong Nam, Kyoung-Seok Oh, Ju-Hee Bang, Yoojin Jeong, Sea Young Choo, Su In Lee, Hyo Jung Kim, Jeesun Yoon, Tae-Yong Kim, Do-Youn Oh. Anti-tumor effects of fadraciclib, CDK2/9 inhibitor, in biliary tract cancer [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 5711.

Abstract 1824: Hexadecanoic acid mediated cell cycle arrest involving inhibition of cell migration

Abstract The Caribbean region accounts for the highest incidence and mortality rates of prostate cancer in the world. This warrants the need for targeted therapy as currently the most prevalent and effective option include chemotherapeutic drugs. Previous studies showed that an enriched sample extracted from the Jamaican Round Leaf yellow yam exhibited antiproliferative effects on DU145 prostate cancer cells. Based on the results obtained, this present study aims at exploring the underlying mechanism of action as well as the antimetastatic activities that the yam derived hexadecanoic acid may exhibit. Flow cytometry analysis was used to assess cell cycle arrest exhibited by hexadecanoic acid on the hormone insensitive DU145 prostate cancer cells. The western blot analysis was then utilized to determine the effects of the hexadecanoic acid enriched sample on cyclins in the cells. Transwell migration assays were then used to evaluate cell metastatic capabilities. Hexadecanoic acid enriched sample induced G1 S cell cycle arrest in the hormone insensitive prostate cancer cell line. Western blot assays also indicated that cell death was possibly induced through the cell cycle regulatory protein, cyclin D1 as its expression was downregulated in the treatment groups. Additionally, the observed metastatic inhibition of prostate cancer cells due to the action of the hexadecanoic acid in the wound healing assay may be attributed to a mechanism involving cyclin D1 as studies have indicated that abnormal Ccnd1 Cdk4 expression promotes tumor growth and metastasis. These results are substantial as it indicates a mechanistic pathway through which hexadecanoic acid is able to cause antiproliferative effects through a cell cycle arrest of hormone insensitive prostate cancer cell lines. Citation Format: Sasha-gay A. Wright, William Aiken, Helen Asemota, Kimberly Foster, Rupika Delgoda. Hexadecanoic acid mediated cell cycle arrest involving inhibition of cell migration [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 1824.

“Keep on ROCKIn”: Repurposed ROCK inhibitors to boost corneal endothelial regeneration

The global shortage of corneal endothelial graft tissue necessitates the exploration of alternative therapeutic strategies. Rho-associated protein kinase inhibitors (ROCKi), recognized for their regenerative potential in cardiology, oncology, and neurology, have shown promise in corneal endothelial regeneration. This study investigates the repurposing potential of additional ROCKi compounds. Through screening a self-assembled library of ROCKi on B4G12 corneal endothelial cells, we evaluated their dose-dependent effects on proliferation, migration, and toxicity using live-cell imaging. Nine ROCKi candidates significantly enhanced B4G12 proliferation compared to the basal growth rate. These candidates were further assessed for their potential to accelerate wound closure as another indicator for tissue regeneration capacity, with most demonstrating notable efficacy. To assess the potential impact of candidate ROCKi on key corneal endothelial cell markers related to cell proliferation, leaky tight junctions and ion efflux capacity, we analyzed the protein expression of cyclin E1, CDK2, p16, ZO-1 and Na+/K+-ATPase, respectively. Immunocytochemistry and western blot analysis confirmed the preservation of corneal endothelial markers post-treatment with ROCKi hits. However, notable cytoplasm enlargement and nuclear fragmentation were detected after the treatment with SR-3677 and Thiazovivin, indicating possible cellular stress. In compared parameters, Chroman-1 at a concentration of 10 nM outperformed other ROCKi, requiring significantly 1000-fold lower effective concentration than established ROCKi Y-27632 and Fasudil. Altogether, this study underscores the potential of repurposing ROCKi for treating corneal endothelial dysfunctions, offering a viable alternative to conventional grafting methods, and highlights Chroman-1 as a promising candidate structure for hit-to-lead development.