CDK4 Research Articles

Comparison of transcriptome profiles of mesenchymal stem cells derived from umbilical cord and bone marrow of giant panda (Ailuropoda melanoleuca)

Mesenchymal stem cells (MSCs) have pluripotent differentiation ability and play an important role in human clinical cell therapy. While, the research on MSCs in endangered wild animals is extremely rare. In our previous studies, the bone marrow mesenchymal stem cells (bmMSCs) and umbilical cord mesenchymal stem cells (ucMSCs) of giant panda (Ailuropoda melanoleuca) were successfully isolated. We aimed to characterize the differences in gene expression profiles between these two types of MSCs using RNA sequencing (RNA-Seq) and to determine which potential pathways are involved in functional regulation. In total, 1079 significantly differentially expressed genes (DEGs) were identified, of which 478 genes were upregulated and 601 genes were downregulated. The significantly enriched Gene Ontology (GO) terms mainly contained cell adhesion, biological adhesion, intracellular signal transduction, molecular function regulator, Ras protein signal transduction, small GTPase mediated signal transduction, and regulation of Rho protein signal transduction. The most enrichment pathways of DEGs enriched in Kyoto Encyclopedia of Genes Genomes (KEGG) were PI3K-AKT signaling pathway, Rap1 signaling pathway, MAPK signaling pathway, Hippo signaling pathway, Wnt signaling pathway, cGMP-PKG signaling pathway and Signaling pathways regulating pluripotency of stem cells. In addition, quantitative real time polymerase chain reaction (qRT-PCR) showed that the AKT3, CDK2, MAPK3, mTOR, PI3K and PTK2 genes associated with PI3K-AKT pathway were highly expressed (P < 0.01), and Caspase-3 was low expressed (P < 0.05) in ucMSCs group when compared with bmMSCs. After treatment with the PI3K inhibitor LY294002, genes AKT3, CDK2, MAPK3, mTOR, and PTK2 were significantly decreased in ucMSCs (P < 0.01), and Caspase-3 was significantly up regulated (P < 0.001). In conclusion, we for the first time compared and analyzed the transcriptome profiles of giant panda ucMSCs and bmMSCs, and found the PI3K-AKT pathway was highly activated and might be a key signaling pathway in the ucMSCs regulation. This study will be beneficial for the research on MSCs proliferation regulation and differentiation of giant pandas in the future, and lay the foundation for MSCs application and clinical therapy for endangered wild animals.

Downregulation of miR-885-5p Promotes NF-κB Pathway Activation and Immune Recruitment in Cutaneous Lupus Erythematosus

Cutaneous lupus erythematosus (CLE) has a specific microRNA expression profile. MiR-885-5p has been found to be downregulated in the epidermis of CLE lesions; however, its biological role in the disease has not been studied. In this study, we show that miR-885-5p is markedly reduced in CLE keratinocytes (KCs) with IFN-α and UVB being strong miR-885-5p regulators invitro. Microarray expression profiling of anti‒miR-885-5p‒transfected KCs identified PSMB5 as a direct target. Specific inhibition of miR-885-5p increased epidermal proliferation by modulating keratin 16 gene K16, BIRC5, TP63, and CDK4 proliferative genes and promoted NF-κB signaling pathway in human primary KCs by increasing IκBα degradation. Silencing PSMB5 rescued the effect of miR-885-5p inhibition, indicating that miR-885-5p regulates proliferation and NF-κB activation by targeting PSMB5 in KCs. In addition, inhibition of miR-885-5p increased the ability of KCs to attract leukocytes in a PSMB5-independent manner. We identified TRAF1 as another direct target, and its silencing reduced leukocyte migration. Collectively, our findings suggest that UVB and IFN-ɑ downregulate miR-885-5p in CLE KCs, leading to epidermal inflammation by NF-κB activity enhancement and proliferation through PSMB5 and immune recruitment through TRAF1. Our data indicate that miR-885-5p is a potential therapeutic target in CLE.

Deficiency of nitrogen but not phosphorus triggers the life cycle transition of the dinoflagellate Scrippsiella acuminata from vegetative growth to resting cyst formation

Nitrogen (N) and phosphorus (P) are essential elements for algal growth. When N and P are deficient, dinoflagellates will take a series of measures to achieve population continuation including formation of resting cysts, an important ecological strategy of dinoflagellates that plays a key role in the initiation and termination of harmful algal blooms (HABs). How the deficiency of N and P affects algal growth and cyst formation has been investigated in some dinoflagellate species, but how it affects the life cycle transition in dinoflagellates has been poorly understood. In this study, we further explored the effect of N and P deficiency on the algal growth and resting cyst production in the cosmopolitan HABs-causing species Scrippsiella acuminata via refining the N and P concentration gradients. Further, we tracked the expression patterns of one CyclinB and one CDK1 genes of S. acuminata at different growth stages under three deficiency concentrations (1/1000 dilutions of N, P, and both N and P). The results suggest that N deficiency always triggered the cyst formation but P deficiency mainly inhibited the vegetative growth instead of inducing cyst formation. We also observed the highest cyst production when S. acuminata was cultured in the f/2-Si medium that was a one-thousandth dilution of N and P (N∼ 0.882 μM; P∼ 0.0362 μM). Our results for the expressions of CyclinB and CDK1 were well consistent with the results of algal growth and cyst formation at different deficiencies of N and P in terms of that higher expressions of these two genes were corresponding to higher rates of vegetative cell growth, while their expressions in resting cysts maintained to be moderate but significantly lower than that in fast-growing vegetative cells. Although we are still not sure whether the changing expressions of the two genes did regulate the transition of life cycle (i.e. cyst formation), or happened as parallels to the expressions of other truly regulating genes, our observations are surely inspirational for further investigations on the genetic regulation of life cycle transition in dinoflagellates. Our work will provide clues to probe the physiological and molecular mechanisms underlying the nutrient deficiency-induced alternation between life cycle stages in dinoflagellates.

NADPH Oxidase 2 Has a Crucial Role in Cell Cycle Progression of Esophageal Squamous Cell Carcinoma.

NADPH oxidases (NOXs) are transmembrane proteins that generate reactive oxygen species. Recent studies have reported that NOXs are involved in tumor progression in various cancers. However, the expression and role of NOX2 in esophageal squamous cell carcinoma (ESCC) remain unclear. This study aimed to clarify the pathophysiologic role of NOX2 in patients with ESCC and cell lines. Two human ESCC cell lines (TE5 and KYSE170) were used for NOX2 transfection experiments, and the effects on cell proliferation, cell cycle, cell motility, and cell survival were analyzed. An mRNA microarray analysis was also performed to assess gene expression profiles. Additionally, NOX2 immunohistochemistry was performed on 130 primary ESCC tumor samples to assess the prognostic value of NOX2 in patients with ESCC. NOX2 depletion significantly inhibited cell proliferation with the G0/G1 arrest and resulted in apoptosis in two cell lines. Microarray analysis revealed a strong relationship between NOX2 gene expression and the signaling pathway of cell cycle regulation by the B-cell translocation gene 2 (BTG2) family, including BTG2, CCNE2, E2F1, and CDK2 genes. Immunohistochemical staining revealed that high NOX2 protein expression was significantly associated with deeper tumor invasion and selected as one of the independent prognostic factors associated with the 5-year OS rate in patients with ESCC. NOX2 expression in ESCC cells affects tumorigenesis, especially cell cycle progression via the BTG2-related signaling pathway, as well as the prognosis of patients with ESCC. NOX2 may be a novel biomarker and therapeutic target for ESCC.

Association of CDK6 gene polymorphisms with Behcet's disease in a Han Chinese population

Cyclin-dependent kinases 4/6 (CDK4/6) and D1-type cyclins (CCND1) can regulate the pro-inflammatory functions of various cytokines during the inflammatory response. This study investigated the association between CDK4/6-CCND1 variants and susceptibility in patients with Behcet's disease (BD). This case-control study enrolled 542 patients with BD and 754 healthy controls. Fourteen tagged single nucleotide polymorphisms (tag SNPs) of the CDK4/6-CCND1 gene were genotyped using the Sequenom MassARRAY system and iPLEX® Pro assay. The results indicated that the frequency of the CDK6 rs2282983 TT genotype was higher in the BD group than the control group (Pc = 0.040, OR = 1.408, 95% CI = 1.124–1.765), and CDK6 rs2282983 CT and rs42034 AG were negatively associated with BD (Pc = 3.647 × 10−4, OR = 0.598, 95% CI = 0.471–0.758; Pc = 0.039, OR = 0.626, 95% CI = 0.459–0.852, respectively). Furthermore, statistical analysis showed that CDK6 rs2282983 TT and CT genotypes were significantly associated with skin lesions in patients with BD (Pc = 0.042, OR = 1.436, 95% CI = 1.130–1.824; Pc = 0.001, OR = 0.594, 95% CI = 0.461–0.764, respectively). This study suggests that the CDK6 loci rs2282983 and rs42034 might confer genetic susceptibility to BD in a Han Chinese population, which could provide new insights into the pathogenesis of BD.

Metformin and Gegen Qinlian Decoction boost islet α-cell proliferation of the STZ induced diabetic rats

BackgroundThe traditional Chinese medicine Gegen Qinlian Decoction (GQD), as well as metformin, had been reported with anti-diabetic effects in clinical practice.ObjectiveTo verify whether these two medicines effectively ameliorate hyperglycemia caused by deficiency of islet β-cell mass which occurs in both type 1 and type 2 diabetes.MethodsSD rats were injected with a single dose of STZ (55 mg/kg) to induce β-cell destruction. The rats were then divided into control, diabetes, GQD and metformin group. GQD and metformin groups were administered with GQD extract or metformin for 6 weeks. The islet α-cell or β-cell mass changes were tested by immunohistochemical and immunofluorescent staining. The potential targets and mechanisms of GQD and metformin on cell proliferation were tested using in silico network pharmacology. Real-time PCR was performed to test the expression of islet cells related genes and targets related genes.ResultsBoth GQD and metformin did not significantly reduce the FBG level caused by β-cell mass reduction, but alleviated liver and pancreas histopathology. Both GQD and metformin did not change the insulin positive cell mass but increased α-cell proliferation of the diabetic rats. Gene expression analysis showed that GQD and metformin significantly increased the targets gene cyclin-dependent kinase 4 (Cdk4) and insulin receptor substrate (Irs1) level.ConclusionThis research indicates that GQD and metformin significantly increased the α-cell proliferation of β-cell deficiency induced diabetic rats by restoring Cdk4 and Irs1 gene expression.

Synthesis and Evaluation of Some New 4H-Pyran Derivatives as Antioxidant, Antibacterial and Anti-HCT-116 Cells of CRC, with Molecular Docking, Antiproliferative, Apoptotic and ADME Investigations.

Colorectal cancer oncogenesis is linked to dysbiosis, oxidative stress and overexpression of CDK2. The 4H-pyran scaffold is considered an antitumoral, antibacterial and antioxidant lead as well as a CDK2 inhibitor. Herein, certain 4H-pyran derivatives were evaluated as antibacterial, antioxidant and cytotoxic agents against HCT-116 cells. Derivatives 4g and 4j inhibited all the tested Gram-positive isolates, except for B. cereus (ATCC 14579), with lower IC50 values (µM) than ampicillin. In addition, 4g and 4j demonstrated the strongest DPPH scavenging and reducing potencies, with 4j being more efficient than BHT. In cell viability assays, 4d and 4k suppressed the proliferation of HCT-116 cells, with the lowest IC50 values being 75.1 and 85.88 µM, respectively. The results of molecular docking simulations of 4d and 4k, inhibitory kinase assays against CDK2, along with determination of CDK2 protein concentration and the expression level of CDK2 gene in the lysates of HCT-116 treated cells, suggested that these analogues blocked the proliferation of HCT-116 cells by inhibiting kinase activity and downregulating expression levels of CDK2 protein and gene. Moreover, 4d and 4k were found to induce apoptosis in HCT-116 cells via activation of the caspase-3 gene. Lastly, compounds 4g, 4j, 4d and 4k were predicted to comply with Lipinski’s rule of five, and they are expected to possess excellent physiochemical and pharmacokinetic properties suitable for in vivo bioavailability, as predicted by the SwissADME web tool.

P387: UNRAVELING LEUKEMIC STEM CELLS MITOCHONDRIAL DEPENDENCY IN PEDIATRIC ACUTE MYELOID LEUKEMIA

Background: Relapse still represents an unsolved problem for children with acute myeloid leukemia (AML), therefore more effective therapeutic strategies are needed for a complete blasts eradication. Leukemic stem cells (LSCs) have been proposed as the therapy-resistant reservoir of cells responsible for failure of antiproliferative chemotherapy and disease recurrence in AML due to their self-renewal capacity and quiescence allowing evasion from chemotherapy. Thus, the identification of LSCs-specific vulnerabilities is needed for more efficacious treatments. Aims: We aim to dissect novel LSCs characteristics to unravel mechanisms of chemoresistance and disease recurrence, and characterize biological properties of LSCs that can be targeted to completely eradicate AML. Methods: To study LSCs derived from primary pediatric AML samples, we based on reactive oxygen species (ROS) content since it was shown that functionally defined LSCs have relatively low levels of ROS. We isolated the lowest 20% of cells fluorescing with CellROX probe (ROS low) and the highest 20% (ROS high) by cell sorting, and analyzed cell cycle, engraftment in mice and RNA sequencing to identify deregulated pathways. Results: After isolating ROS low and ROS high cells, we first assessed classical stemness features. By colony-forming assay, we observed that ROS low and ROS high cells have similar colony-forming capacity, but only ROS low cells preserve this capacity after a second replating whereas the ROS high population does not (p=0.002). By BrdU assay, we found that ROS low cells are relatively dormant, being more in G0/G1 cell cycle phase (41.9%) than the ROS high counterpart (7.9%, p=0.001), also confirmed by CFSE staining, revealing that ROS low cells proliferate more slowly (at day 4, 31% undivided cells in ROS low vs 8% in ROS high). According to our previously published data, we confirmed that ROS low cells express higher levels of CDK6-AS1 (RQ=1.38) and CDK6 genes (RQ=2.69; RQ=1 for ROS high) and that, being more quiescent, are characterized by lower levels of mitochondrial membrane potential by TMRE and lower content of mitochondrial ROS, byproducts of mitochondrial activity, by MitoSOX staining. We also verified that ROS low cells have a lower mitochondrial mass by MitoTracker and TOMM20 staining (mitochondria to nucleus ratio 1.18 vs 1.35 for ROS high, p=0.0005), suggesting that mitochondrial mass and functions might be involved in quiescent cells properties. However, engraftment experiments did not reveal different capacity of ROS low or ROS high cells in recreating leukemia in NSG mice, neither in terms of human CD45+ cells in mice peripheral blood (23.3% vs 41.9% respectively, p=0.12) nor bone marrow infiltration (73% vs 84.8%, p=0.44) at 8 weeks post-engraftment, opening for further dissection of the presence of LSCs within ROS high cells. By RNA sequencing, we found that the top 150 differentially expressed genes between ROS low and ROS high cells are involved in focal adhesion (logP=-13.51), regulation of hemopoiesis (logP=-12.31), cell response to stress (logP=-8.73) and cytokine production (logP=-8.67). Further analyses will confirm the role of these pathways in maintaining ROS low LSCs. Moreover, we identified 5 differentially expressed surface antigens that are under investigation as putative ROS low LSCs markers. Summary/Conclusion: In conclusion, the ROS low strategy permits to identify a quiescent AML subpopulation with peculiar mitochondrial features and transcriptome allowing to pinpoint novel molecular pathways that might be targeted to enhance LSCs clearance.

Abstract 1174: Exploration of MDM2 gene amplification, co-mutation status, and prognosis in solid tumors

Abstract Loss of p53 tumor suppressor function is critical for many cancers and is achieved by mutation in ~50% of tumors, but may occur through additional mechanisms. MDM2 is an E3 ubiquitin ligase whose primary function is to inhibit p53 activity by impeding p53 transcriptional activity, promoting nuclear export, and inducing p53 degradation. The use of investigational MDM2 inhibitors such as milademetan (RAIN-32) may provide a therapeutic strategy for select tumors with wildtype (WT) TP53. Gene amplification (amp) represents a mechanism for cancer cells to upregulate expression of critical cancer related genes and can serve as a predictive biomarker for targeted therapies. Previous work established a gene copy number (CN) threshold of ≥ 12 for the MANTRA-2 basket study of milademetan in advanced solid tumors (Tirunagaru et al., AACR-NCI-EORTC 2021). We used publicly available AACR-GENIE data and The Cancer Genome Atlas (TCGA) to explore MDM2 amp in relation to TP53 status and other genetic biomarkers across solid tumors. MDM2 amp occurs in most cancer types, with the most frequent rates of MDM2 amp occurring in sarcomas, bladder, hepatobiliary, non-small cell lung cancer, gastric/esophageal, breast, and melanoma cancers. MDM2 was mutually exclusive of TP53 mutations at CN ≥ 12 with OR = 0.21 (p = 5.3 x 10-8). Tumors with WT TP53 and MDM2 CN ≥ 12 comprised 1.19% of all solid tumors in the TCGA (n=9,090) and 1.22% in the AACR-Genie dataset (n=42,125). The MDM2 and CDK4 genes are located on chromosome 12q13-15 and these genes are frequently co-amplified in LPS and other sarcomas; however, MDM2 and CDK4 co-amp is variable in other tumors with less frequent co-amp occurring in NSCLC and melanoma and rare co-amp occurring in bladder, breast cancer and gastro-esophageal tumors, particularly at MDM2 CN ≥ 12. We also evaluated co-mutations in known oncogenes (filtered for oncogenic variants using cancerhotspots.org) in tumors with MDM2 CN ≥ 4. Co-alteration of MDM2 amp in this group of tumors was &amp;lt;15% for all MDM2 amp tumors and did not vary significantly with increasing MDM2 CN. OS survival analysis revealed that prognosis for patients with all levels of MDM2 amp was worse compared to non-amp tumors and OS at MDM2 CN ≥ 12 [41.7 months(m)] was similar to tumors harboring TP53 mutations (55.0m) as compared to patients with tumors harboring WT TP53 and MDM2 CN &amp;lt;12 (118.8m). In conclusion, MDM2 amp was mutually exclusive of TP53 mutations at CN ≥ 12 and occurred in 1.2% of all cancers and occurred across multiple tumor types, supportive of a tumor agnostic therapeutic strategy for milademetan. CDK4 was not co-amplified with MDM2 in many tumor types and oncogenic alterations occurred infrequently with MDM2 amp. MDM2 amp was associated with a poor prognosis consistent with an unmet need in these cancer patients. The MANTRA-2 trial evaluating milademetan in patients with solid tumors harboring MDM2 CN ≥ 12 and WT TP53 is currently enrolling patients (NCT05012397). Citation Format: Vijaya G. Tirunagaru, Feng Xu, Robert C. Doebele. Exploration of MDM2 gene amplification, co-mutation status, and prognosis in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1174.

Abstract 2151: Identification and proteogenomic characterization of novel lung adenocarcinoma subtypes with therapeutic relevance

Abstract Lung adenocarcinoma (LUAD) is one of the most common cancer types with various available treatment modalities. However, better biomarkers of response are still needed for further improving precision medicine. Therefore, a robust LUAD subtyping can substantially aid in determining the most effective therapies that target subtype-specific vulnerabilities. In this study, we integrated multiple datasets: (i) the full 509 LUAD patient cohort from The Cancer Genome Atlas (TCGA) project, (ii) cancer vulnerability data in LUAD cell lines from the Broad Institute’s DependencyMap, and (iii) proteomic data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) LUAD patients. Using these datasets, we identified 5 expression subtypes (S1-S5) with unique proteogenomic and dependency profiles that increased the resolution of previously defined subtypes (Proximal Inflammatory [PI]; Proximal Proliferative [PP]; and Terminal Respiratory Unit [TRU]). S4-associated cell lines exhibited specific vulnerability to CDK6 and CDK6-cyclin D3 complex gene, CCND3. S3 was characterized by dependency on CDK4, immune-related expression patterns, and altered MET signaling. Experimental validation showed that S3-associated cell lines responded to MET inhibitors, which also led to increased PD-L1 expression. Finally, we identified a small set of biomarkers for S3 and S4 that could be used in the clinic to classify patients into our therapeutically relevant subtypes. Overall, our lung adenocarcinoma expression subtypes, especially S3 that represents 20% of LUAD patients and S4 that represents 25% of LUAD patients, and their biomarkers could help identify patients likely to respond to CDK4/6, MET, or PD-L1 inhibitors, potentially improving patient outcome. Citation Format: Whijae Roh, Yifat Geffen, Mendy Miller, Shankara Anand, Jaegil Kim, David Heiman, Matthew Meyerson, Peter Laird, Andrew Cherniack, NCI CCG Tumor Molecular Pathology (TMP) Analysis Working Group, Gad Getz. Identification and proteogenomic characterization of novel lung adenocarcinoma subtypes with therapeutic relevance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2151.

Abstract 3108: Preclinical studies of chemotherapies and targeted therapies to address gastric cancer disparities

Abstract Gastric cancer (GC) is one of the most common malignant tumors of the digestive system, and its prognosis remains poor because most tumors were detected in late stages and the 5-year survival is less than 5%. The Cancer Genome Atlas (TCGA) study found that 70% of all GCs have mutations that can be targeted with existing drugs, yet only four molecularly-guided therapies have been approved. GCs have strong disparities in incidence, mortality and survival that disproportionately affect different populations of color. No large-scale cancer genomic research has been carried out in U.S. minority populations. GC molecular data from U.S. minority residents is crucial to improve outcomes, as genomic phenotypes have been associated with differential prognosis and response to treatments. To our knowledge, minority patient-derived models have not been used to test GC therapies. In this study, several organoids lines from minority patients were established and used as an in vitro platform for screening effective therapies. After genomic data analyses, six minority patient-derived organoids with or without cell cycle regulator or PI3K/AKT/mTOR pathway gene alterations were tested for responses to first-line therapies, targeted drugs and combination treatments. Among the 24 PDX models established, the efficacy study of PDX model PDX015 which has amplifications of CDK and AKT genes showed the targeting therapy (a CDKi) can effectively prolong overall survival of mice bearing xenograft treated with the CDKi (60 days), compared with those mice in control group (22 days). In addition, an AKT inhibitor was found significantly inhibited tumor growth in CDKi-resistant mice treated with the CDKi/AKTi combination. In conclusion, investigating the efficacy of targeted therapies in these U.S. minorities will empower precision medicine applications in GC patients and improve GC treatment outcomes. Minority-patient derived tumor models have great potential in preclinical studies of chemotherapies and targeted therapies to address gastric cancer disparities. Citation Format: Hongyong Zhang, Ana Patricia Estrada-Florez, Paul Lott, Ted William Toal, Alexa Morales-Arana, Alma Poceros-Coba, UCaMP Consortium, Ai-Hong Ma, Nicole Britney Halmai, Luis Carvajal-Carmona. Preclinical studies of chemotherapies and targeted therapies to address gastric cancer disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3108.

Abstract 5649: Bromodomain inhibition reduces CDK6 expression and reverses CDK4/6 inhibitor resistance in ER+ breast cancer

Abstract Introduction: CDK4/6 inhibitor (CDK4/6i) resistance is a pressing clinical problem for patients with ER+ breast cancer who are treated with CDK4/6 inhibitors combined with hormonal therapy. Our previous work identified CDK6 overexpression as a critical determinant of acquired resistance to CDK4/6i. Bromodomain and extra-terminal domain (BET) family proteins (BRD2, BRD3, BRD4, and BRDT) play important roles in regulating the expression of oncogenes in multiple cancer types. BET inhibitors (BETi), such as JQ1, have been extensively studied for the treatment of cancers. It has been shown that BRD4 is recruited to the CDK6 gene promoter and that BET inhibition reduces CDK6 mRNA and protein expression. In this study, we investigated the potential of BET inhibition to reverse CDK4/6i resistance of ER+ breast cancer. Methods: By long-term exposure to increasing concentrations of a CDK4/6i, we developed MCF7 and T47D ER+ breast cancer cell lines with acquired resistance to several FDA-approved CDK4/6 inhibitors demonstrating marked CDK6 overexpression. Cell viability and survival assays were performed to assess the in vitro efficacy of BETi. Flow cytometry was performed to evaluate cell cycle alterations as well as cell death. Gene expression was analyzed by Western blotting and qPCR to examine cell cycle-related molecular changes. Markers of apoptosis were also evaluated by Western blotting. CDK4/6i-resistant ER+ tumors were established in immunodeficient mouse models to explore the in vivo efficacy of BETi. Results: Our data showed that JQ1 significantly reduced cell viability and survival of CDK4/6i-resistant ER+ breast cancer cells in the presence of palbociclib, a commonly used CDK4/6i for the treatment of metastatic ER+ HER2- breast cancer. JQ1 also induced marked G1 cell cycle arrest and apoptosis in CDK4/6 inhibitor-resistant MCF7 and T47D cell lines in a time and dose-dependent manner. Analyses of key molecular mediators of cell cycle progression identified CDK6 and cyclin D1 as the major targets of JQ1 in CDK4/6i-resistant cells. Furthermore, we demonstrated that JQ1 suppressed CDK6 expression at the mRNA level. Importantly, we determined that growth suppression and killing of CDK4/6i-resistant cells by BETi were largely the consequence of CDK6 downregulation by BET inhibition. We have also established CDK4/6i-resistant xenografts successfully from MCF7 cells to test the in vivo efficacy of BETi in reversing CDK4/6 inhibitor resistance. Conclusions: Our results suggest that BET inhibition has potent efficacy in reversing acquired CDK4/6i resistance of ER+ breast cancer, mainly through its inhibitory regulation of CDK6. BET inhibitors are therefore promising candidate therapeutic agents for the treatment of CDK4/6i-resistant ER+ breast cancer. Citation Format: Renyan Liu, Constantia Pantelidou, Heta Jadhav, Geoffrey Shapiro. Bromodomain inhibition reduces CDK6 expression and reverses CDK4/6 inhibitor resistance in ER+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5649.

Abstract 2568: ARTS-021 is a potent and selective CDK2 inhibitor that demonstrates anti-cancer activity in preclinical cancer models with CCNE1 amplification

Abstract Cyclin E1 amplification is prevalent in cancers with high unmet medical need such as high grade serous ovarian cancer, stomach cancer and esophageal cancer. Cancer cell lines with high expression level of cyclin E1 gene exhibited profound sensitivity to CDK2 gene depletion, suggesting CDK2 selective inhibitors have the potential to treat patients harboring Cyclin E1 alterations. Here we report the development and preclinical characterization of ARTS-021, an orally bioavailable small molecule CDK2 selective inhibitor. In enzymatic assay against a panel of different CDKs, ARTS-021 potently inhibits the activity of CDK2/CyclinE1 complex with 50% inhibitory concentration (IC50) in the sub-nanomolar range, displaying superior selectivity against other CDKs (CDK1,4,6,7,9). Very low hit rate (S(10)=0.022, at 1uM) is observed when ARTS-021 is assessed in whole Kinome profiling, further revealing the selectivity of this compound. ARTS-021 also demonstrates potent CDK2 inhibition and selectivity against other CDK family members (CDK1,4,6,9) in a series of cellular assays. Consistent with genetic CDK2 dependence data, cell lines with CCNE1 amplification are particularly sensitive to ARTS-021. Double nanomolar ARTS-021 inhibits Rb phosphorylation and blocks G1/S transition, leading to cell growth arrest specifically in CCNE1 amplified cell lines. This CDK2 dependent anti-tumor activity is further validated in vivo, where daily ARTS-021 administration leads to tumor stasis in CCNE1 amplified but not wild type xenograft models. Taken together, these data demonstrate ARTS-021 as a promising CDK2 selective inhibitor with strong potential towards the development of effective therapies for CCNE1 altered cancer. Citation Format: jiaqi Liang, Dai Cheng, Yali Guo, xiaobin zhang, Xipan Liu, Jia Fu, Qiang Ding, Wen Xiong, Fang Li, Yaoyu Chen, Qing Sheng. ARTS-021 is a potent and selective CDK2 inhibitor that demonstrates anti-cancer activity in preclinical cancer models with CCNE1 amplification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2568.

Clinical significance of next generation sequencing (NGS) in Chinese patients with relapsed/refractory Wilm’s tumor.

e22014 Background: Medical advances have been greatly improved in the five-year survival rate for Wilms’ tumor, but the survival is still dismal for relapsed/refractory (R/R) patients. In the present study, we aimed to investigate the genomic profiling and the incidence of germline cancer susceptibility mutationsof Chinese patients with R/R WT. Methods: Next-generation sequencing (NGS) of tumor specimens with matched blood samples obtained from R/R pediatric WT in Sun Yat-Sen University Cancer Center were used in a CAP-certified laboratory (Simcere Diagnostic Co, Ltd). Clinical information including age, gender and tumor histology were collected. Somatic and germline mutations including single nucleotide variants (SNV), insertion-deletion variants (Indels), copy number variations (CNVs) and fusion, as well as tumor mutational burden (TMB) were analyzed. Results: Nineteen patients with R/R WT were enrolled in this study, including 10 males and 9 females, with a median age of 4 years old (range, 0-14). All of the pathology was identified as favorable histology WT (FHWT). Somatic mutations were detected in 84.21% (16/19) of patients, and CNVs accounted for the majority of the total somatic aberrations, followed by SNVs. The most common somatic mutations were identified in the following genes, CTNNB1 , ERBB2, MCL1 , CDK2 and MDM4. Of the top 3 frequently mutated genes, the mutation frequency and their most common mutation type were CTNNB1, 26%, in-frame deletion, missense mutation; ERBB2 21%, missense mutation, amplification; MCL1 21%, amplification. It is worth noting that MCL1 mutation has never been reported in Chinese R/R WT patients before, which may be a potential valuable mechanism. Germline mutations were detected in 89.47% (17/19) of patients, and CNVs accounted for the majority of the total germline aberrations, followed by SNVs. The most common germline mutations were identified in the following genes, MCL1, RET, ALK, EPCAM, and FANCA. Of the top frequently mutated genes frequency and most common mutation type were MCL1 21%, amplification; RET 21% missense mutation, multiple mutations. There are also some suspected clinically pathogenic germline mutation of genes CDK4 , ERBB2, and MDM4 . All the mutation type were amplification. Notable cancer driver mutations were also characterized, such as the amplifications of ERBB2 / MET/EGFR / FGFR1, and the point mutations of CHEK2/FBXW7/NF1. The median TMB was 1.47 (range, 0-7.8). Conclusions: Overall, clinical and new results were detected in 14 (73.68%) in Chinese R/R WT patients. We discovered some driver genes mutations which potentially sensitive to the corresponding targeted drugs in Chinese R/R WT patients. Large sample size was needed to provide a better understanding of molecular features in R/R WT patients to achieve precision medicine.

Telomere as a Therapeutic Target in Dedifferentiated Liposarcoma.

Simple SummaryDDLPS is a non-lipogenic tumor with aggressive clinical behavior. DDLPS patients have limited therapeutic options, especially those with retroperitoneal tumors, and their outcome remains largely unsatisfactory, highlighting the need for novel treatment strategies. Gene expression analysis carried out in clinical samples from primary retroperitoneal DDLPS indicated that these tumors highly express genes involved in telomere maintenance, the cellular process that guarantees immortality to tumor cells. In this study, we evaluated the effect of RHPS4, a molecule able to alter telomere by binding to telomeric structures called G-quadruplexes, in patient-derived DDLPS cell lines. Exposure to RHPS4 induced DNA damage and decreased cell proliferation and migration, thus suggesting telomere as a novel target and G-quadruplex binders as innovative therapeutic agents in DDLPS.Background: Well-differentiated (WD)/dedifferentiated (DD) liposarcoma (LPS) accounts for ~60% of retroperitoneal sarcomas. WDLPS and DDLPS divergently evolve from a common precursor and are both marked by the amplification of the 12q13–q15 region, leading to the abnormal expression of MDM2, CDK4, and HMGA2 genes. DDLPS is a non-lipogenic disease associated with aggressive clinical behavior. Patients have limited therapeutic options, especially for advanced disease, and their outcome remains largely unsatisfactory. This evidence underlines the need for identifying and validating DDLPS-specific actionable targets to design novel biology-driven therapies. Methods: Following gene expression profiling of DDLPS clinical specimens, we observed the up-regulation of “telomere maintenance” (TMM) pathways in paired DD and WD components of DDLPS. Considering the relevance of TMM for LPS onset and progression, the activity of a telomeric G-quadruplex binder (RHPS4) was assessed in DDLPS patient-derived cell lines. Results: Equitoxic concentrations of RHPS4 in DDLPS cells altered telomeric c-circle levels, induced DNA damage, and resulted in the accumulation of γ-H2AX-stained micronuclei. This evidence was paralleled by an RHPS4-mediated reduction of in vitro cell migration and induction of apoptosis/autophagy. Conclusions: Our findings support telomere as an intriguing therapeutic target in DDLPS and suggest G-quadruplex binders as innovative therapeutic agents.

Identification of Enhancer RNA CDK6-AS1 as a Potential Novel Prognostic Biomarker in Gastric Cancer.

Background: This study aimed to confirm the role of enhancer RNAs (eRNAs) in gastric cancer and their clinical utility. Methods: We used Cox survival and relevance analysis to identify the candidate eRNAs in gastric cancer and performed Gene Ontology and Reactome pathway enrichment to determine the potential functions of eRNAs. Correlation between eRNA, tumor-infiltrating immune cells, and drug sensitivity was then analyzed. Results: CDK6-AS1, a long non-coding RNA cyclin-dependent kinase 6, may serve as a poor potential prognostic biomarker candidate in gastric cancer with a positive correlation with its target gene CDK6. The low CDK6-AS1 expression group showed more frequent mutated driver genes than the high expression group. Moreover, CDK6-AS1 is involved in a key oncogenic pathway of the cell cycle and RNA transcription. CDK6-AS1 also shows dysregulations and associations with prognosis at the pan-cancer level. This eRNA may also be associated with immune cell infiltration and drug sensitivity. Conclusion: CDK6-AS1 may be a potential prognostic biomarker and chemotherapeutic drug sensitivity predictor in gastric cancer.

Histomorphological changes and molecular mechanisms underlying the ameliorative effect of resveratrol on the liver of silver nanoparticles-exposed rats

ABSTRACT Exposure to the deleterious effects of silver nanoparticles (AgNPs) is inevitable due to their wide use in medicine and daily life. The current study aimed to delineate the histomorphological changes and the molecular mechanisms underlying the ameliorative effect of Resveratrol (RSV) on rats’ livers exposed to AgNPs. Fifty healthy adult male Wistar albino rats were divided into four groups: control, AgNPs-exposed, RSV-treated after AgNPs exposure, and recovery groups. Liver sections were examined by light and electron microscopes, and immunohistochemistry was performed for detection of activated caspase3 and TNFα. Serum ALT and AST, plasma levels of TNFα, IL-6, GSH and SOD were measured. mRNA expression of SIRT1, ADORA3, PAI, CDK1, Nrf2 and NFκB genes in liver tissue homogenate was performed using qRT-PCR. AgNPs-exposure for 28 days caused marked liver tissue damage with trapping in hepatocytes and Kupffer cells, while RSV treatment ameliorated liver ultrastructure and function. Our results clarified the molecular basis of RSV ameliorative effect on liver tissue by significant upregulation of SIRT1-NrF2 signaling pathway with increased levels of the antioxidant GSH and SOD, which represent the antioxidant effect of RSV. Significant upregulation of the protective ADORA3 with downregulation of the proinflammatory PAI-1 and NFκB mRNA expression levels besides decreased plasma levels of TNFα, IL-6 and decreased immunoexpression of TNFα in liver tissue, represent the anti-inflammatory effect of RSV. In addition, decreased immunoexpression of caspase3 and downregulation of CDK1 expression, represent its antiapoptotic effect. In conclusion: RSV ameliorates AgNPs-induced liver damage by antioxidant, anti-inflammatory and antiapoptotic effects. Abbreviations: AgNPs: Silver nanoparticles, RSV: Resveratrol, ROS: Reactive oxygen species, ESR: Electron spin resonance, DMPO: 5,5-Dimethyl-1-pyrroline-N-oxide, H2O2: Hydrogen peroxide, SOD: Superoxide dismutase, CAT: Catalase, GPx: Glutathione peroxidase, MPTP: Methyl-4-phenyl-1.2.3.6-tetrahydropyridine, MDA: Malondialdehyde, TNF: Tumor necrosis factor, GSH: Glutathione, Nrf2: Nuclear factor-erythroid 2-related factor 2, ARE: Antioxidant response elements, KEAP1: Kelch-1ike ECH-associated protein l, AMPK: AMP-activated protein kinase, HO-1: Heme oxygenase-1, NF-κB: Nuclear factor-kappa B, SIRT1: Sirtuins, FOXO: Forkhead box, UCP2: Uncoupling protein 2, STZ: Streptozotocin nicotinamide, HSC: hepatic stellate cells, ECM: extracellular matrix.

High Expression of the Lysosomal Protease Cathepsin D Confers Better Prognosis in Neuroblastoma Patients by Contrasting EGF-Induced Neuroblastoma Cell Growth.

Neuroblastoma is a malignant extracranial solid tumor arising from the sympathoadrenal lineage of the neural crest and is often associated with N-MYC amplification. Cathepsin D has been associated with chemoresistance in N-MYC-overexpressing neuroblastomas. Increased EGFR expression also has been associated with the aggressive behavior of neuroblastomas. This work aimed to understand the mechanisms linking EGFR stimulation and cathepsin D expression with neuroblastoma progression and prognosis. Gene correlation analysis in pediatric neuroblastoma patients revealed that individuals bearing a high EGFR transcript level have a good prognosis only when CTSD (the gene coding for the lysosomal protease Cathepsin D, CD) is highly expressed. Low CTSD expression was associated with poor clinical outcome. CTSD expression was negatively correlated with CCNB2, CCNA2, CDK1 and CDK6 genes involved in cell cycle division. We investigated the biochemical pathways downstream to EGFR stimulation in human SH-SY5Y neuroblastoma cells engineered for overexpressing or silencing of CD expression. Cathepsin D overexpression decreased the proliferative potential of neuroblastoma cells through downregulation of the pro-oncogenic MAPK signaling pathway. EGFR stimulation downregulated cathepsin D expression, thus favoring cell cycle division. Our data suggest that chemotherapeutics that inhibit the EGFR pathway, along with stimulators of cathepsin D synthesis and activity, could benefit neuroblastoma prognosis.

CDK6-PI3K signaling axis is an efficient target for attenuating ABCB1/P-gp mediated multi-drug resistance (MDR) in cancer cells

BackgroundMultidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1/P-gp) is a major cause of cancer chemotherapy failure, but the regulation mechanisms are largely unknown.MethodsBased on single gene knockout, we studied the regulation of CDK6-PI3K axis on ABCB1-mediated MDR in human cancer cells. CRISPR/Cas9 technique was performed in KB-C2 cells to knockout cdk6 or cdk4 gene. Western blot, RT-PCR and transcriptome analysis were performed to investigate target gene deletion and expression of critical signaling factors. The effect of cdk4 or cdk6 deficiency on cell apoptosis and the cell cycle was analyzed using flow cytometry. In vivo studies were performed to study the sensitivity of KB-C2 tumors to doxorubicin, tumor growth and metastasis.ResultsDeficiency of cdk6 led to remarkable downregulation of ABCB1 expression and reversal of ABCB1-mediated MDR. Transcriptomic analysis revealed that CDK6 knockout regulated a series of signaling factors, among them, PI3K 110α and 110β, KRAS and MAPK10 were downregulated, and FOS-promoting cell autophagy and CXCL1-regulating multiple factors were upregulated. Notably, PI3K 110α/110β deficiency in-return downregulated CDK6 and the CDK6-PI3K axis synergizes in regulating ABCB1 expression, which strengthened the regulation of ABCB1 over single regulation by either CDK6 or PI3K 110α/110β. High frequency of alternative splicing (AS) of premature ABCB1 mRNA induced by CDK6, CDK4 or PI3K 110α/110β level change was confirmed to alter the ABCB1 level, among them 10 common skipped exon (SE) events were found. In vivo experiments demonstrated that loss of cdk6 remarkably increased the sensitivity of KB-C2 tumors to doxorubicin by increasing drug accumulation of the tumors, resulting in remarkable inhibition of tumor growth and metastasis, as well as KB-C2 survival in the nude mice.ConclusionsCDK6-PI3K as a new target signaling axis to reverse ABCB1-mediated MDR is reported for the first time in cancers. Pathways leading to inhibition of cancer cell proliferation were revealed to be accompanied by CDK6 deficiency.

Genetic risk factors in melanoma etiopathogenesis and the role of genetic counseling: A concise review

Melanoma is a highly aggressive cancer originating from melanocytes. Its etiopathogenesis is strongly related to genetic, epigenetic, and environmental factors. Melanomas encountered in clinical practice are predominantly sporadic, whereas hereditary melanomas account for approximately 10% of the cases. Hereditary melanomas mainly develop due to mutations in the cyclin-dependent kinase 2A (CDKN2A) gene, which encodes two tumor suppressor proteins involved in the cell cycle regulation. CDKN2A, along with CDK4, TERT, and POT1 genes, are high-risk genes for melanoma. Among the genes that carry a moderate risk are MC1R and MITF, whose protein products are involved in melanin synthesis. The environment also contributes to the development of melanoma. Patients at risk of melanoma should be offered genetic counseling to discuss genetic testing options and the importance of skin UV protection, avoidance of sun exposure, and regular preventive dermatological examinations. Although cancer screening cannot prevent the development of the disease, it allows for early diagnosis when the survival rate is the highest.