Upregulation Of CDKN1A Research Articles

Involvement of multiple epigenetic mechanisms by altered DNA methylation from the early stage of renal carcinogenesis before proliferative lesion formation upon repeated administration of ochratoxin A

Ochratoxin A (OTA) is a rat renal carcinogen that induces karyomegaly and micronuclei in proximal tubular epithelial cells (PTECs). We previously performed comprehensive gene profiling of alterations in promoter-region methylation and gene expression in PTECs of rats treated with OTA for 13 weeks. The OTA-specific gene profile was obtained by excluding genes showing expression changes similar to those upon treatment with 3-chloro-1,2-propanediol, a renal carcinogen not inducing karyomegaly. In this study, we validated the candidate genes using methylated DNA enrichment PCR and real-time RT-PCR, and identified Gen1, Anxa3, Cdkn1a, and Osm as genes showing OTA-specific epigenetic changes. These genes and related molecules were subjected to gene expression and immunohistochemical analyses in the PTECs of rats treated with OTA, other renal carcinogens, or non-carcinogenic renal toxicants for 4 or 13 weeks. Cdkn1a upregulation and increase of p21WAF1/CIP1+ karyomegalic PTECs were observed with OTA, matching the findings associated with micronucleus-inducing carcinogens. This suggested that the increase of p21WAF1/CIP1+ karyomegalic PTECs is linked to micronucleus formation, which in turn accelerates chromosomal instability. The upregulation of Cdkn1a-related genes with OTA suggests the acquisition of a senescence-associated secretory phenotype, which promotes the establishment of a carcinogenic environment. Meanwhile, OTA specifically caused a decrease of GEN1+ PTECs reflecting Gen1 downregulation and an increase of ANXA3+ PTECs reflecting Anxa3 upregulation, as well as Osm upregulation. OTA may efficiently disrupt pathways for repairing the DNA double-strand breaks that it itself causes, via Gen1 downregulation, and enhance cell proliferation through the upregulation of Anxa3 and Osm. This may exacerbate the chromosomal instability from the early stage of OTA-induced renal carcinogenesis before proliferative lesions form. OTA may cause renal carcinogenesis involving multiple epigenetic mechanisms.

Safety, pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of KT-253, a targeted protein degrader of MDM2, in patients with relapsed/refractory (R/R) solid tumors, lymphoma, high grade myeloid malignancies and acute lymphoblastic leukemia (ALL).

3084 Background: The tumor suppressor p53 is mutated in approximately 50% of cancers. In those cancers with wild-type p53, its activity is controlled by mouse double minute 2 (MDM2), an E3 ligase that tags p53 for degradation. KT-253 is a novel, highly potent heterobifunctional MDM2 degrader that upregulates p53 activity and overcomes the p53-MDM2 feedback loop, resulting in >200-fold higher potency compared to MDM2 inhibitors. In preclinical PDX models of sensitive p53WT solid tumors, AML and ALL, KT-253 robustly activates p53, induces apoptosis, and results in tumor regressions with every 3-week dosing. The ability to rapidly induce an acute apoptotic response and dose intermittently may result in a therapeutic index that has eluded previous MDM2 targeting agents. Methods: The ongoing open-label Phase 1 study evaluates safety, PK, PD and preliminary efficacy of IV KT-253 administered on Day 1 of 21-day cycles. Patients (pts) with advanced (≥2 prior therapies) solid tumors (ST)/lymphomas (Arm A) and relapsed/refractory (R/R) high grade myeloid malignancies (AML, high/very-high risk myelodysplastic syndrome (MDS), MDS/myeloproliferative neoplasms) and ALL (Arm B) are eligible. Blood samples are collected for KT-253 PK/PD analyses. Results: As of 26 January 2024, 18 pts have been treated, 13 in Arm A dose levels (A: DL) 1-4 and 5 in Arm B dose levels (B: DL) 1-2, with mean number of 3 and 2 doses, respectively. The most common solid tumor types were Merkel cell carcinoma ((MCC) n=3), adenoid cystic carcinoma ((ACC) n=2) and uveal melanoma (n=2). Arm B included 5 pts with R/R AML. The median age was 61 years (yrs) (range 42, 81) in Arm A and 66 yrs (range 57, 70) in Arm B. The most common adverse events (AEs) in > 20% of pts included nausea, fatigue, headache, and vomiting. There was 1 DLT of AEs leading to discontinuation that included Grade (G) 2 nausea and fatigue in A: DL4. There were no neutropenia or thrombocytopenia AEs in either arm. KT-253 related SAEs included G3 hypotension in a pt with decreased oral intake (A: DL1). Overall best response: 1 CR (B: DL2, AML); 2 PRs (A: DL1, MCC; B: DL1, AML); 3 SD (A: DL1, fibromyxoid sarcoma; DL2, ACC; DL3, renal cell cancer). PD data from A: DL1-4 and B: DL1-2 demonstrated rapid upregulation of plasma GDF-15 protein and upregulation of CDKN1A and PHLDA3 mRNA levels in blood. KT-253 demonstrated dose-dependent increase in plasma exposure with levels approximating efficacious doses. Conclusions: KT-253 results in potent upregulation of p53-dependent biomarkers and has demonstrated early signs of anti-tumor activity including objective responses in MCC and AML at doses that are well tolerated. Dose escalation is ongoing at DL4 in Arm A and at DL2 in Arm B and analyses from additional pts will be presented at the meeting. Clinical trial information: NCT05775406 .

Exploring CDKN1A Upregulation Mechanisms: Insights into Cell Cycle Arrest Induced by NC2603 Curcumin Analog in MCF-7 Breast Cancer Cells.

Breast cancer stands out as one of the most prevalent malignancies worldwide, necessitating a nuanced understanding of its molecular underpinnings for effective treatment. Hormone receptors in breast cancer cells substantially influence treatment strategies, dictating therapeutic approaches in clinical settings, serving as a guide for drug development, and aiming to enhance treatment specificity and efficacy. Natural compounds, such as curcumin, offer a diverse array of chemical structures with promising therapeutic potential. Despite curcumin's benefits, challenges like poor solubility and rapid metabolism have spurred the exploration of analogs. Here, we evaluated the efficacy of the curcumin analog NC2603 to induce cell cycle arrest in MCF-7 breast cancer cells and explored its molecular mechanisms. Our findings reveal potent inhibition of cell viability (IC50 = 5.6 μM) and greater specificity than doxorubicin toward MCF-7 vs. non-cancer HaCaT cells. Transcriptome analysis identified 12,055 modulated genes, most notably upregulation of GADD45A and downregulation of ESR1, implicating CDKN1A-mediated regulation of proliferation and cell cycle genes. We hypothesize that the curcumin analog by inducing GADD45A expression and repressing ESR1, triggers the expression of CDKN1A, which in turn downregulates the expression of many important genes of proliferation and the cell cycle. These insights advance our understanding of curcumin analogs' therapeutic potential, highlighting not just their role in treatment, but also the molecular pathways involved in their activity toward breast cancer cells.

Abstract B016: Novel curcumin analogues as therapies for endometrial cancer with TP53 mutations

Abstract The protein p53 is designated as the guardian of the genome because it is the principal tumor suppressor and functions to surveil the genome, enforce cell cycle checkpoints, and trigger apoptosis when genomic instability is beyond repair. Mutations in TP53 are found in >50% of human tumors and in 25% of the deadliest endometrial malignancies. Yet, few studies have targeted the mutant protein to restore its wild type (WT) tumor suppressor functions. Curcumin, a turmeric derivative, has been shown to help return WT function to mutant p53. However, curcumin is not bioavailable to most of the body because of its water insolubility, hydrophobic nature and rapid metabolization. Our laboratory is testing novel, more bioavailable analogs of curcumin including HO-3867 and AKT-100. In these studies, we tested these agents in preclinical models of serous endometrial cancer to assess their effects on cell growth and p53-dependent signaling. We used CyQUANT cell proliferation assay to identify IC50 values for HO-3867 and AKT-100 in ECC-1 and KLE endometrial cancer cell lines. We performed RNA sequencing to elucidate mechanisms of action and verified p53 expression changes via Western blotting. IC50 values ranged from 100 nanomolar (AKT-100) to low micromolar (HO-3867). RNA sequencing data revealed the re-enforcement of cell cycle checkpoints including the upregulation of CDKN1A (p21) and GADD45 (Gadd45). Proliferative signaling downstream of ATM, an important protein kinase stimulating cell growth, was downregulated by both agents. Western blotting confirmed the classic negative feedback loop of p53, where total p53 levels decreased after drug treatment. Taken together, our findings show that AKT-100 and HO-3867 are able to restore WT tumor suppressor function in otherwise oncogenic mutant p53 by reducing cell replication and promoting apoptosis. Thus, curcumin analogues such as AKT-100 and HO-3867 show promise in the treatment of a subset of endometrial tumors expressing mutant p53 by returning WT p53 functionality, and future studies will move to in vivo analyses of curcumin analogue function. Citation Format: Kimberly K. Leslie, Alex Goss, Jamie Padilla, Lane E. Smith. Novel curcumin analogues as therapies for endometrial cancer with TP53 mutations [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B016.

Pre-Clinical Efficacy of CDK7 Inhibitor-Based Combinations in Cellular Models of Advanced Myeloproliferative Neoplasms (MPN) Transformed to AML

Advanced MPN are characterized by driver mutations in JAK2, c-MPL or calreticulin (CALR) gene, with hyperactive JAK-STAT5/3 and NFkB signaling. Co-occurrence of mutations in chromatin/transcriptional regulators, including TET2, ASXL1, EZH2, SRSF2, RUNX1 and TP53, creates the dysregulated epigenome/transcriptome/proteome that inhibits differentiation and induces leukemia transformation (sAML) and therapy-refractoriness in MPN with excess blasts (> 5% in PB) or MPN transformed to AML (sAML). Treatment with JAK inhibitor (JAKi), e.g., ruxolitinib, venetoclax or hypomethylating agents alone or in combination are ineffective in improving the poor survival in MPN-sAML. CDK7 is the catalytic component of the general transcription factor TFIIH recruited with RNA-pol II (RNAP2) to transcription start-sites to initiate mRNA transcription. By phosphorylating CDK9 and negative transcript elongation factors, CDK7 also enables productive transcript elongation of oncogenes involved in growth and survival. In MPN-sAML, these include RUNX1, Bcl-xL, MCL1, CDK4/6, PIM1, and MYC. By phosphorylating CDK1/2/4/6, CDK7 also induces cell cycle. RNA-Seq analysis in BEAT AML study showed significantly higher CDK7 expression in MPN-sAML compared to normal progenitor cells. DepMap data involving CRISPR-mediated gene-knockouts show CDK7 as a dependency in the MPN-sAML SET2 and HEL cell lines. Present studies demonstrate that treatment with ATP-competitive, covalent CDK7 inhibitors (CDK7i) SY-1365, and clinical grade SY-5609, dose-dependently (20 to 250 nM) increased % G1 while reducing the % of cell-cycle S phase SET2 and HEL cells. CDK7i treatment induced in vitro loss of viability in these cells, as well as in patient-derived (PD), CD34+ MPN-sAML but not in normal CD34+ progenitor cells. CRISPR-mediated CDK7 depletion (over 85%) in HEL cells significantly reduced SY-5609-mediated loss of viability. Importantly, pre-treatment with SY-5609 significantly increased sensitivity to ruxolitinib-induced loss of viability in JAKi-resistant SET2 and HEL cells, suggesting that CDK7 inhibition could overcome resistance to JAKi in MPN-sAML cells. RNA-Seq analysis conducted after SY-5609 treatment (100 nM for 16 hours) showed significant negative-enrichment of the gene-sets of MYC and E2F targets, cell-cycle checkpoints and of protein translation initiation/elongation, with log2 fold-decline in the mRNA levels of MYC, MYB, PLK1/4, KIT, CDK6, AURKA, PIM1 and CCND1, but upregulation of CDKN1A and HEXIM1. Following SY-5609 treatment of HEL cells, mass spectrometry demonstrated significant log2 fold-decline in c-Myc, c-Myb, RUNX1, PLK1, PIM1, but increase in p21, CASP9, BAD, DAP and TGFβ1 protein levels. CyTOF analysis demonstrated that SY-5609 treatment markedly reduced c-Myc, RUNX1 and MCL1, while increasing TP53, p21, and cleaved PARP proteins levels in MPN-sAML stem-progenitor cells defined by high expression of CD34, CLEC12A, CD123, CD99 but low expression of CD11b. Co-treatment with SY-5609 and ruxolitinib induced synergistic loss of viability in HEL, SET2 and PD MPN-sAML cells (n = 5) (delta synergy scores > 1.0 by the ZIP method). A CRISPR screen in SET2 and HEL cells targeting epigenetic regulators revealed significant log2 fold-decline in gRNA targeting BRD4, CBP and p300, highlighting them as dependencies. Consistent with this, co-treatment with SY-5609 and the BETi OTX015 was synergistically lethal in SET2, HEL, mouse MPN (JAK2-V617F plus TP53 loss) cells and PD MPN-sAML cells (n=5). SY-5609 treatment also exerted synergistic lethality with the BETi pelabresib or BD2-selective BETi ABBV-744 or the CBP/p300 inhibitor GNE-049 in MPN-sAML cells. In the xenograft model of HEL-Luc/GFP cells in NSG mice, monotherapy with SY-5609 (1.5 or 3.0 mg/kg/day, by oral gavage), vs vehicle control, significantly reduced the MPN-sAML burden and improved survival, without causing toxicity. Additionally, compared to each drug or vehicle control, co-treatment with SY-5609 and OTX015 (30 mg/kg/day by oral gavage) reduced more MPN-sAML burden and significantly improved survival in a HEL-Luc/GFP xenograft model without inducing toxicity. These findings demonstrate promising preclinical activity of CDK7 inhibition against the cellular models of MPN-sAML, supporting the rationale to further evaluate in vivo efficacy of CDK7i-based combinations against advanced MPN with excess blasts or MPN-sAML.

SBDSR126T rescues survival of sbds -/- zebrafish in a dose-dependent manner independently of Tp53.

Defects in ribosomal biogenesis profoundly affect organismal development and cellular function, and these ribosomopathies produce a variety of phenotypes. One ribosomopathy, Shwachman-Diamond syndrome (SDS) is characterized by neutropenia, pancreatic exocrine insufficiency, and skeletal anomalies. SDS results from biallelic mutations in SBDS, which encodes a ribosome assembly factor. Some individuals express a missense mutation, SBDS R126T , along with the common K62X mutation. We reported that the sbds-null zebrafish phenocopies much of SDS. We further showed activation of Tp53-dependent pathways before the fish died during the larval stage. Here, we expressed SBDS R126T as a transgene in the sbds -/- background. We showed that one copy of the SBDS R126T transgene permitted the establishment of maternal zygotic sbds-null fish which produced defective embryos with cdkn1a up-regulation, a Tp53 target involved in cell cycle arrest. None survived beyond 3 dpf. However, two copies of the transgene resulted in normal development and lifespan. Surprisingly, neutropenia persisted. The surviving fish displayed suppression of female sex differentiation, a stress response in zebrafish. To evaluate the role of Tp53 in the pathogenesis of sbds -/- fish phenotype, we bred the fish with a DNA binding deficient allele, tp53 M214K Expression of the loss-of-function tp53 M214K did not rescue neutropenia or survival in sbds-null zebrafish. Increased expression of cdkn1a was abrogated in the tp53 M214K/M214K ;sbds -/- fish. We conclude that the amount of SBDSR126T protein is important for development, inactivation of Tp53 fails to rescue neutropenia or survival in the sbds-null background, and cdkn1a up-regulation was dependent on WT tp53 We hypothesize that additional pathways are involved in the pathophysiology of SDS.

Oxidative stress induced by Pollonein-LAAO, a new L-amino acid oxidase from Bothrops moojeni venom, prompts prostate tumor spheroid cell death and impairs the cellular invasion process in vitro

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies.

Deletion of eif6 Partially Rescues Survival, but Not Neutropenia, of Zebrafish Model of Shwachman-Diamond Syndrome: The Roles of Sbds and Eif6 in Perturbing Lipid Metabolism

Introduction: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by pancreatic insufficiency, skeletal defects, neutropenia, and an increased risk of myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML). Biallelic mutations in SBDS account for ~90% of patients. SBDS interacts physically with EFL1 to release EIF6 from the pre-60S ribosomal subunit and promote the assembly of the mature 80S ribosome in the cytoplasm. A subset of SDS patients acquire del(20q), resulting in loss of EIF6. Additionally, somatic heterozygous EIF6 mutations have been found in SDS patients. SDS patients harboring del(20q) and EIF6 mutations are linked to a lower risk of developing MDS/AML when compared to other SDS patients. Together these factors point to a potential mechanism whereby a reduction in EIF6 levels may lead to a suppression in ribosome biogenesis defects. Methods and Results: We have created sbds knockout (KO) zebrafish strains that phenocopy the human syndrome with stunted growth, neutropenia and atrophy in the pancreas, liver, and intestine. Unlike the Sbds-null mice which die in early embryogenesis, sbds-null fish survive to early juvenile stages. The sbds KO fish display an accumulation of Eif6 in liver and intestine. Interestingly, we also found an accumulation of EIF6 in SDS patient-derived lymphoblasts. EIF6 has been described as a key regulator of metabolism, specifically stimulating glycolytic and fatty acid synthesis. Lipid metabolomics of sbds KO fish show a decrease in free fatty acids and phosphatidylcholine. Expression of several genes critical in lipid metabolism (srebp1, fasn and pparg) is upregulated in sbds KO fish. To study the role of Eif6 in the pathophysiology of SDS, we created an eif6 KO zebrafish with an insertion of one base pair resulting in a premature stop codon. By immunoblotting we demonstrated that mutants did not display Eif6 by 6 days post fertilization (dpf), and heterozygous fish displayed less protein than wildtype siblings. This new strain showed early mortality (~10 dpf), neutropenia, tp53 pathway activation, with a pronounced upregulation of cdkn1a as early as 5 dpf. RNA-Seq data of eif6 KO fish at 5 dpf showed a total of 2225 differentially expressed genes (DEGs): 668 downregulated and 1557 upregulated. These DEGs are involved in two main gene expression pathways: ribosome biogenesis and oxidative phosphorylation. We also observed a dysregulation of lipid metabolism markers in the eif6 KO fish. Furthermore, we bred the sbds KO with the eif6 KO, we observed a partial rescue on survival only in sbds-/- with one copy of eif6(eif6+/-) but failed to rescue their neutropenia. Conclusions: Our organismal models of sbds or eif6 deletion provide new insights into the pathophysiology of human SDS: 1) SBDS affects lipid metabolism possibly due to an accumulation of EIF6, 2) Loss of eif6 affects development/survival at an earlier stage than loss of sbds, and 3) Loss of either sbds or eif6 markedly upregulates cdkn1a, which is downstream of tp53. Interestingly, Eif6 partially rescues survival of sbds-null organisms, but only in the haploinsufficient state. EIF6 may offer a promising target for a novel therapeutic strategy in SDS.

Antiviral Response and HIV-1 Inhibition in Sickle Cell Disease

BACKGROUND: A number of host restriction factors controls HIV-1 infection. While many of these factors have been investigated in vitro, their role in controlling HIV-1 infection in vivo remains to be elucidated. Here, we investigate a naturally occurring HIV-1 restriction in Sickle cell disease (SCD), a hereditary disorder characterized by sickle cell hemoglobin polymerization and red blood cells hemolysis. We previously showed that SCD PBMCs express higher levels of ferroportin (FPN), and that reduced intracellular iron leads to the upregulation of antiviral restriction factors. Previously, HIV-1 infection was shown to inversely correlate with the expression levels of transferrin receptor (TFR), a marker of reduced intracellular iron. Iron chelation also potently inhibits HIV-1 replication as shown by us and others. Thus, iron metabolism is intrinsically connected to the HIV-1 infection and disease progression. Our study in vitro suggests that SCD environment leads to the antiviral state of T cells and macrophages, and suppression of HIV-1 infection. Here we analyzed HIV infection of SCD mice using mouse adapted EcoHIV and compared antiviral factors in SCD mouse spleenocytes and human SCD PBMCs. METHODS: The study was approved by Howard University IRB and IACUC committees. Townes SCD mice carrying human normal α (hα) and sickle hemoglobin beta (βS) genes and control Townes mice carrying human normal hα and βA genes were used in the study. EcoHIV virus obtained from David Volsky (Albert Einstein University) was constructed in pNL4-3 vector with the substitution of gp120 with the envelope-coding gp80 region from ectopic MLV. Whole blood samples were obtained from 9 SCD patients and 9 healthy individuals. RNA Seq analysis was conducted on Illumina® NextSeq 500 and the data were mapped and analyzed using Dragen software. Normalized gene counts were used for gene set enrichment analysis (GSEA) and Gene ontology (GO) analysis. RNA expression was measured by quantitative RT-PCR (Roche). Protein expression in mouse splenocytes was determined by ELISA. RESULTS: Infection of control mice with EcoHIV virus, which contained MLV envelope and was able to infect mouse cells, led to robust viral replication in spleen at 7 days post infection, as detected by the expression of HIV-1 Nef mRNA (Fig. 1) and Gag mRNA. We also detected HIV-1 p24 in splenic tissue. Comparing to the control, SCD mice showed significantly reduced EcoHIV replication both at the levels of RNA expression (Figs 1) and p24 production. GSEA analysis against the 72 genes that included all known HIV-1 restriction factors revealed strong enrichment of these genes in the human activated SCD PBMCs compared to the activated control PBMCs (Fig.2). These genes included APOBEC3A, HMOX1, CH25H, IFIT3, CDKN1A, APOBEC3B, IFITM3, SAMHD1 and TSPO. GSEA analysis also showed upregulation of several pathogen-associated molecular patterns sensing genes including NOD1, NLRC4, TRL7 and TLR9 in human PBMCS. In SCD mice splenocytes, we detected overexpression of FPN, and transferrin receptor (Tfr) genes indicating upregulation of iron metabolism similar to previously observed in human SCD PBMCs. In mouse splenocytes we detected upregulation of CDKN1A, HMOX1, IFITM3, CH25H and TSPO mRNAs. Protein expression analysis showed upregulation of FPN, TFR, HO-1, APOBEC, CH25H, IFIT3 and TSPO in SCD mouse splenocytes. CONCLUSION: Together, these data reveal, for the first time, that HIV-1 infection is suppressed in vivo in SCD mouse infected with the mouse-adapted EcoHIV. SCD mouse splenocytes expressed higher levels of FPN and TFR, and exhibited higher expression of antiviral factor, which were also observed in human SCD PBMCs. The observed upregulation of similar sets of antiviral factors in SCD PBCMs and SCD mice splenocytes suggests that these factors upregulation might be driven by the SCD-related processes such as hemolysis. Taken together, our findings point to a novel mechanism of upregulation of antiviral factors mediated by SCD that included induction of antiviral, heme- and iron- regulatory pathways. ACKNOWLEDGEMENTS: We thank Dr. David Volsky for the gift of EcoHIV and the George Washington University SPH Genomics Core for library preparation and sequencing. This work was supported by NIH grants 1R01HL125005, 5U54MD007597, 1P30AI117970-06 and 1SC1HL150685. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-25. EXPLOITING EPIGENETIC DYSREGULATION IN ISOCITRATE DEHYDROGENASE MUTANT GLIOMA VIA THE USE OF HISTONE DEACETYLASE INHIBITORS

Abstract Isocitrate dehydrogenase 1/2 mutations (IDHmut) define a unique subtype of glioma that exhibits profound epigenetic dysregulation through DNA and histone hypermethylation. Though IDHmutgliomas are less aggressive than their IDH wildtype (IDHwt) counterparts, these tumors are still incurable and are invariably fatal. New treatments are therefore needed. Histone deacetylase (HDAC) enzymes compose a class of epigenetic drug targets already used in the clinic to treat other malignancies, but their potential against gliomas, specifically IDHmutgliomas, have not been extensively studied. We previously showed that IDHwt gliomas are more sensitive than IDHwt gliomas to the cytotoxic and antiproliferative effects mediated by the FDA-approved HDAC inhibitor panobinostat, and that this sensitivity was associated with a greater increase in histone acetylation in IDHmut glioma cultures. Follow-up RNA-Seq showed that panobinostat more greatly affected gene expression in IDHmut glioma cells, with 1999 significant differentially expressed genes showing a 10% or greater change in gene expression in IDHmut cells compared to 388 genes for IDHwt counterparts. This included the known HDAC target and cell-cycle inhibitor CDKN1A, with panobinostat causing a 1.9-fold greater upregulation of CDKN1A in IDHmut cells than IDHwt cells (p= 0.045). In immunocompromised mice intracranially engrafted with IDHmut or IDHwt glioma, 15 mg/kg intraperitoneally-administered panobinostat was detectable after 6 hours within the intracerebral tumors via mass spectrometry at a concentration of 52.4 ng/g (150.1 nM), and extended the survival of IDHmut glioma-engrafted mice (Median: 4 days; Mean: 9.4 days), but not IDHwt glioma-engrafted mice (IDHmut: logrank p= 0.044; IDHwt: logrank p= 0.18). These results suggest that HDAC inhibition via panobinostat may be an effective therapy to treat IDHmutglioma patients. Ongoing studies, including ChIP-Seq, will determine specific sites of histone acetylation and HDACs that are responsible for the greater sensitivity of IDHmut gliomas to HDAC inhibition.

A novel compound EPIC-0412 reverses temozolomide resistance via inhibiting DNA repair/MGMT in glioblastoma.

Temozolomide (TMZ) resistance has become an important obstacle affecting its therapeutic benefits. O6-methylguanine DNA methyltransferase (MGMT) is primarily responsible for the TMZ resistance in Glioblastoma multiforme (GBM) patients. In addition, active DNA damage repair pathways can also lead to TMZ resistance. Here, we reported a novel small-molecule inhibitor EPIC-0412 that improved the therapeutic efficacy of TMZ by inhibiting the DNA damage repair pathway and MGMT in GBM via epigenetic pathways. The small-molecule compound EPIC-0412 was obtained through high-throughput screening. RNA immunoprecipitation (RIP), chromatin isolation by RNA purification (ChIRP), and chromatin immunoprecipitation (ChIP) assays were used to verify the effect of EPIC-0412. Co-immunoprecipitation (Co-IP) was used to elucidate the interactions of transcription factors at the MGMT promoter region. Animal experiments using a mouse model were performed to verify the efficacy of EPIC-0412 in sensitizing GBM cells to TMZ. EPIC-0412 physically interrupts the binding of HOTAIR and EZH2, leading to the upregulation of CDKN1A and BBC3, causing cell cycle arrest and apoptosis in GBM cells. EPIC-0412 inhibits DNA damage response in GBM cells through the p21-E2F1 DNA damage repair axis. EPIC-0412 epigenetically silences MGMT through its interaction with the ATF3-p-p65-HADC1 axis at the MGMT promoter region. The application of EPIC-0412 restored the TMZ sensitivity in GBM in vivo experiments. This study discovered a small-molecule inhibitor EPIC-0412, which enhanced the chemotherapeutic effect of TMZ by acting on the p21-E2F1 DNA damage repair axis and ATF3-p-p65-MGMT axis, providing evidence for combining epigenetic drugs to increase the sensitization toward TMZ in GBM patients.

Negative regulation of CDKN1A by the histone methyltransferase EHMT2 for cell growth in colorectal cancer

The epigenetic regulation of oncogenes and tumor suppressor genes by histone methyltransferases is an important process for colon cancer growth and metastasis. Although various epigenetic modifiers have been recognized as attractive therapeutic targets for colon cancer treatment, alternative epigenetic regulation in colon cancer for reducing side effects and increasing the effectiveness of treatments has not been thoroughly explored. In this study, we identified CDKN1A as a direct target for EHMT2 by RNA-sequencing and found increased growth suppression via upregulation of CDKN1A by EHMT2 knockdown. In addition, using a 3-dimensional culture system for spheroid formation with an ultralow attachment plate, we confirmed EHMT2-related growth suppression and CDKN1A regulation. Thus, we suggest that EHMT2 may be a therapeutic target for colon cancer treatment, and an EHMT2 inhibitor should be developed for the effective treatment of colon cancer.

MiR-6077 promotes cisplatin/pemetrexed resistance in lung adenocarcinoma via CDKN1A/cell cycle arrest and KEAP1/ferroptosis pathways

Lung adenocarcinoma (LUAD) is one of the most common malignancies worldwide. Combination chemotherapy with cisplatin (CDDP) plus pemetrexed (PEM) remains the predominant therapeutic regimen; however, chemoresistance greatly limits its curative potential. Here, through CRISPR-Cas9 screening, we identified miR-6077 as a key driver of CDDP/PEM resistance in LUAD. Functional experiments verified that ectopic overexpression of miR-6077 desensitized LUAD cells to CDDP/PEM in both cell lines and patient-derived xenograft models. Through RNA sequencing in cells and single-cell sequencing of samples from patients with CDDP/PEM treatments, we observed CDDP/PEM-induced upregulation of CDKN1A and KEAP1, which in turn activated cell-cycle arrest and ferroptosis, respectively, thus leading to cell death. Through miRNA pull-down, we identified and validated that miR-6077 targets CDKN1A and KEAP1. Furthermore, we demonstrated that miR-6077 protects LUAD cells from cell death induced by CDDP/PEM via CDKN1A-CDK1-mediated cell-cycle arrest and KEAP1-NRF2-SLC7A11/NQO1-mediated ferroptosis, thus resulting in chemoresistance in multiple LUAD cells both in vitro and in vivo. Moreover, we found that GMDS-AS1 and LINC01128 sensitized LUAD cells to CDDP/PEM by sponging miR-6077. Collectively, these results imply the critical role of miR-6077 in LUAD’s sensitivity to CDDP/PEM, thus providing a novel therapeutic strategy for overcoming chemoresistance in clinical practice.

Hyperglycemia Promotes Endothelial Cell Senescence through AQR/PLAU Signaling Axis.

Hyperglycemia is reported to accelerate endothelial cell senescence that contributes to diabetic complications. The underlying mechanism, however, remains elusive. We previously demonstrated AQR as a susceptibility gene for type 2 diabetes mellitus (T2DM) and showed that it was increased in multiple tissues in models with T2DM or metabolic syndrome. This study aimed to investigate the role of AQR in hyperglycemia-induced senescence and its underlying mechanism. Here, we retrieved several datasets of the aging models and found the expression of AQR was increased by high glucose and by aging across species, including C. elegans (whole-body), rat (cardiac tissues), and monkey (blood). we validated the increased AQR expression in senescent human umbilical vein endothelial cells (HUVECs). When overexpressed, AQR promoted the endothelial cell senescence, confirmed by an increased number of cells stained with senescence-associated beta-galactosidase and upregulation of CDKN1A (P21) as well as the prohibited cellular colony formation and G2/M phase arrest. To explore the mechanism by which AQR regulated the cellular senescence, transcriptomic analyses of HUVECs with the overexpression and knockdown of the AQR were performed. We identified 52 co-expressed genes that were enriched, in the terms of plasminogen activation, innate immunity, immunity, and antiviral defense. Among co-expressed genes, PLAU was selected to evaluate its contribution to senescence for its highest strength in the enrichment of the biological process. We demonstrated that the knockdown of PLAU rescued senescence-related phenotypes, endothelial cell activation, and inflammation in models induced by AQR or TNF-α. These findings, for the first time, indicate that AQR/PLAU is a critical signaling axis in the modulation of endothelial cell senescence, revealing a novel link between hyperglycemia and vascular dysfunction. The study may have implications in the prevention of premature vascular aging associated with T2DM.

Salinomycin induces cell cycle arrest and apoptosis and modulates hepatic cytochrome P450 mRNA expression in HepG2/C3a cells

Salinomycin (SAL) is a monocarboxylic polyether ionophore antibiotic isolated from Streptomyces albus. It exhibits an effective antitumor potential against numerous human cancer cells. This study aimed to assess the antiproliferative effects of SAL in human hepatocellular carcinoma HepG2/C3a cell line. We investigated the effects of SAL on cell growth, DNA damage induction, cell cycle changes and apoptosis; and relative changes in expression of cell cycle-related, apoptosis-related, and CYP450 genes. SAL induced cell cycle arrest in the G2/M phase, upregulation of CDKN1A and GADD45A and downregulation of cyclin genes including CCNB1 and CCNA2. SAL effectively suppressed mRNA levels of CTNNB1 gene, an important oncogene that promotes tumorigenesis. The decrease of HepG2/C3A cells’ survival can also be due to downregulation of antiapoptotic BCL-2 expression, thus promoting the induction of apoptosis by SAL. This study also demonstrated the ability of SAL in modulating hepatic cytochrome P450 (CYP) mRNA expression, such that SAL caused the upregulation of CYP1A members and CYP3A5; and downregulation of CYP3A4. Taken together, these data contribute to the understanding of the mechanism of action of SAL, highlighting that metabolizing enzymes modulated by SAL can interfere with chemotherapy treatment and it must be considered in associated treatments.

Elimination of Radiation-Induced Senescence in the Brain Tumor Microenvironment Attenuates Glioblastoma Recurrence.

Glioblastomas (GBM) are routinely treated with ionizing radiation (IR) but inevitably recur and develop therapy resistance. During treatment, the tissue surrounding tumors is also irradiated. IR potently induces senescence, and senescent stromal cells can promote the growth of neighboring tumor cells by secreting factors that create a senescence-associated secretory phenotype (SASP). Here, we carried out transcriptomic and tumorigenicity analyses in irradiated mouse brains to elucidate how radiotherapy-induced senescence of non-neoplastic brain cells promotes tumor growth. Following cranial irradiation, widespread senescence in the brain occurred, with the astrocytic population being particularly susceptible. Irradiated brains showed an altered transcriptomic profile characterized by upregulation of CDKN1A (p21), a key enforcer of senescence, and several SASP factors, including HGF, the ligand of the receptor tyrosine kinase (RTK) Met. Preirradiation of mouse brains increased Met-driven growth and invasiveness of orthotopically implanted glioma cells. Importantly, irradiated p21-/- mouse brains did not exhibit senescence and consequently failed to promote tumor growth. Senescent astrocytes secreted HGF to activate Met in glioma cells and to promote their migration and invasion in vitro, which could be blocked by HGF-neutralizing antibodies or the Met inhibitor crizotinib. Crizotinib also slowed the growth of glioma cells implanted in preirradiated brains. Treatment with the senolytic drug ABT-263 (navitoclax) selectively killed senescent astrocytes in vivo, significantly attenuating growth of glioma cells implanted in preirradiated brains. These results indicate that SASP factors in the irradiated tumor microenvironment drive GBM growth via RTK activation, underscoring the potential utility of adjuvant senolytic therapy for preventing GBM recurrence after radiotherapy. SIGNIFICANCE: This study uncovers mechanisms by which radiotherapy can promote GBM recurrence by inducing senescence in non-neoplastic brain cells, suggesting that senolytic therapy can blunt recurrent GBM growth and aggressiveness.

H3K36 Trimethylation Mediated By SETD2 Regulates Cell Proliferation and Cell Cycle By Modulating BCMA-JNK and c-Myc Pathways in Multiple Myeloma

In addition to genetic aberrations, accumulating evidence indicates that deregulation of histone methyltransferases, such as MMSET, EZH2 and KDM6A, plays crucial roles in the oncogenic transformation and development of multiple myeloma (MM). For example, overexpression of MMSET leading to a global increase in H3K36me2, is believed to be the driving force in the pathogenesis of t (4;14) MM. However, as the histone methyltransferase is responsible for H3K36me3, the role of SETD2 is not been known in myeloma.To explore the possible clinical value of SETD2 in MM, we first examined the gene expression profile from GEO database, which indicated that the SETD2 expression was significantly decreased in MM patients when compared with monoclonal gammopathy of undetermined significance and smoldering multiple myeloma patients (GSE6477). Moreover, the expression of SETD2 decreased with the advanced international staging system stage of MM patients (GSE19784). The Kaplan-Meier analysis showed that low expression of SETD2 was significantly associated with a poor overall survival in MM patients (GSE2658, P<0.05; GSE9782, P<0.001). Thus, our analysis suggests that SETD2 might participate in cancer progression and could become a biomarker for the prognosis of patients with MM.We then investigated the biological role and the underlying mechanism of SETD2 in MM. Firstly, we used lentiviral-mediated RNA interference to knockdown SETD2 (SETD2 KD) in MM cell lines (RPMI8226 and MM.1S). CCK8 and colony-forming assays showed that reduced expression of SETD2 significantly promoted MM cell proliferation and colony growth. BrdU incorporation assay revealed increased DNA synthesis in SETD2 KD MM cells. Then, treatment with JIB-04, a small molecule inhibitor targeting H3K36me3 loss in SETD2 KD MM cells showed that H3K36me3 recovery was capable of reversing the tumor-promoting effect due to SETD2 down-regulation of MM cells in vitro. Moreover, the xenograft growth assay revealed that SETD2 down-regulation facilitated tumor growth and JIB-04 treatment exhibited anti-myeloma activity in vivo. Therefore, we conclude that SETD2 plays an important role in MM maintenance, and inhibition of H3K36me3 shows therapeutic efficacy for MM.To further explore the underlying mechanisms, we performed the RNA-Seq analysis and discovered that low H3K36me3 level was associated with reduced expression of CDKN1A and increased expression of TNFRSF17 (BCMA) and c-Myc in MM cells. The Gene Set Enrichment Analysis (GSEA) revealed that MAPK signaling pathway was enriched in SETD2 knockdown and JIB-04 treated MM cells. Subsequent Western blotting analysis further confirmed that SETD2 KD cells had increased JNK activation, while JIB-04 treated group showed decreased level of p-JNK. To investigate whether BCMA was the directly transcriptional target for H3K36me3, we carried out a dual-luciferase reporter assay in both SETD2 KD and JIB-04 treated MM cells, and confirmed that H3K36me3 inhibited the expression of BCMA through physically interacting with motifs in its promoter. Furthermore, down-regulation of BCMA in SETD2 KD MM cells could lead to a reduction of p-JNK and an up-regulation of CDKN1A, and resulting inhibited cell proliferation and cell cycle progression. Furthermore, blockage of the JNK pathway by its inhibitor SP600125 resulted in significant inhibition of MM cell proliferation induced by SETD2 knockdown. Additionally, suppression of c‐Myc using 10058‐F4 inhibited proliferation, and induced cell cycle arrest as well as CDKN1A expression in SETD2 KD MM cells. These results indicate that BCMA/JNK and c-Myc pathways were involved in STED2 and H3K36me3 mediated cell proliferation in MM.Together, our data delineate that SETD2-dependent H3K36me3 modification plays a critical role in regulation cell proliferation and cell cycle by BCMA-JNK and c-Myc pathways in MM cells. Targeting the SETD2-H3K36me3 pathway represents a promising therapy for MM. DisclosuresNo relevant conflicts of interest to declare.

Induction of apoptosis on ovarian adenocarcinoma cells, A2780 by tricyclohexylphosphanegold (I) mercaptobenzoate derivatives via intrinsic and extrinsic pathways.

Tricyclohexylphosphanegold(I) n-mercaptobenzoate (n = 2, 3, 4) labelled as 1-3 were previously reported to significantly suppress thioredoxin reductase (TrxR) activities towards ovarian cancer cells, A2780, in vitro. Herein, we explored the role of 1-3 for their apoptosis inducing ability against A2780 cells. 1-3 exhibited IC50 values at 1.19 ± 0.03µM, 2.28 ± 0.04μM and 0.78 ± 0.01μM, respectively, compared to cisplatin at26.8 ± 0.15µM. The compounds induced A2780 apoptosis via a caspase-dependent mitochondrion pathway as evidenced by ROS production, cytochrome c release, caspases-3/7, -8, -9 and -10 activation, APAF1 and BAX upregulation as well as BCL2A1 and BCL2 genes' downregulation. In addition, the death mode of 1-3 was also mediated via death receptor extrinsic pathway manifested by FAS, FASL, FADD, and TNFR1 genes' upregulation via Human Rt PCR analysis. In addition, 1-3 significantly caused A2780 arrest at S phase, which was associated with the upregulation of TP53, E2F1, RB1 and CDKN1A upregulation and downregulation of CDK1, CDK4, CDC25A and CDC25C genes. Based on these promising results, these phosphanegold(I) thiolate derivatives could act as feasible candidates for further advanced in vivo ovarian cancer studies to develop novel chemotherapeutic agents derived from metal-based agents.

EHMT1 knockdown induces apoptosis and cell cycle arrest in lung cancer cells by increasing CDKN1A expression.

Dozens of histone methyltransferases have been identified and biochemically characterized, but the pathological roles of their dysfunction in human diseases such as cancer remain largely unclear. Here, we demonstrate the involvement of EHMT1, a histone lysine methyltransferase, in lung cancer. Immunohistochemical analysis indicated that the expression levels of EHMT1 are significantly elevated in human lung carcinomas compared with non‐neoplastic lung tissues. Through gene ontology analysis of RNA‐seq results, we showed that EHMT1 is clearly associated with apoptosis and the cell cycle process. Moreover, FACS analysis and cell growth assays showed that knockdown of EHMT1 induced apoptosis and G1 cell cycle arrest via upregulation of CDKN1A in A549 and H1299 cell lines. Finally, in 3D spheroid culture, compared to control cells, EHMT1 knockdown cells exhibited reduced aggregation of 3D spheroids and clear upregulation of CDKN1A and downregulation of E‐cadherin. Therefore, the results of the present study suggest that EHMT1 plays a critical role in the regulation of cancer cell apoptosis and the cell cycle by modulating CDKN1A expression. Further functional analyses of EHMT1 in the context of human tumorigenesis may aid in the development of novel therapeutic strategies for cancer.

Co-Adjuvant Therapy Efficacy of Catechin and Procyanidin B2 with Docetaxel on Hormone-Related Cancers In Vitro.

Prostate (PC) and breast cancer (BC) are heterogeneous hormonal cancers. Treatment resistance and adverse effects are the main limitations of conventional chemotherapy treatment. The use of sensitizing agents could improve the effectiveness of chemotherapeutic drugs as well as obviate these limitations. This study analyzes the effect of single catechin (CAT), procyanidin B2 (ProB2) treatment as well as the co-adjuvant treatment of each of these compounds with docetaxel (DOCE). We used PC- and BC-derived cell lines (PC3, DU-145, T47D, MCF-7 and MDA-MB-231). The short and long-term pro-apoptotic, anti-proliferative and anti-migratory effects were analyzed. RT-qPCR was used to discover molecular bases of the therapeutic efficacy of these compounds. ProB2 treatment induced a two- to five-fold increase in anti-proliferative and pro-apoptotic effects compared to single DOCE treatment, and also had a more sensitizing effect than DOCE on DU145 cells. Regarding BC cells, ProB2- and CAT-mediated sensitization to DOCE anti-proliferative and pro-apoptotic effects was cell-independent and cell-dependent, respectively. Combined treatment led to high-efficacy effects on MCF-7 cells, which were associated to the up-regulation of CDKN1A, BAX, caspase 9 and E-cadherin mRNA under combined treatment compared to single DOCE treatment. CAT and ProB2 can enhance the efficacy of DOCE therapy on PC and BC cells by the sensitizing mechanism.