Methyltransferase Research Articles

Hepatitis B virus X protein induces p16 gene promoter methylation through upregulation of DNA methylation transferases DNMT1 and DNMT3A.

As a tumor suppressor, p16 can competitively block the cyclin D1-CDK4/6 complex to arrest the cell cycle in the G1 phase. Lack of p16 gene expression can lead to infinite cell proliferation and malignant transformation. To determine whether the hepatitis B virus X protein (HBx) regulates the methylation and expression of the p16 gene. We constructed a eukaryotic expression vector carrying the HBx gene and green fluorescent protein (GFP), and transfected it into HepG2 cells to build cell lines stably expressing GFP and GFP-HBx. The p16 protein level and p16 gene methylation were measured in these cell lines. We further detected the mRNA expression of DNA methyltransferases (DNMTs) 1, 3A and 3B. The DNMT1, DNMT3A and DNMT3B gene promoter sequences were inserted into a reporter vector (pGL3-Basic) to build recombinant vectors, which were then transiently transfected into different cell lines. After 48 h, the luciferase activity was measured. The level of p16 protein in GFP-HBx/HepG2 cells was significantly lower than in HepG2 and GFP/HepG2 cells. The CpG methylation was present in the p16 gene promoter region of GFP-HBx/HepG2 cells. The DNMT1 and DNMT3A mRNA levels in GFP-HBx/HepG2 cells were significantly elevated compared to that in the HepG2 cells (p = 0.0495). The luciferase activity in GFP-HBx/HepG2 cells transfected with the pGL3-DNMT1/3A pro plasmid was significantly higher than in HepG2 and GFP/HepG2 cells (both p < 0.05). The HBx can induce p16 gene promoter methylation and inhibit the expression of p16 in HepG2 cells. This occurs due to HBx activation of DNMT1 and DNMT3A promoters and the induction of p16 promoter methylation, which downregulates the expression of p16 protein.

Balance between autophagy and cell death is maintained by Polycomb-mediated regulation during stem cell differentiation.

Autophagy is a conserved cytoprotective process, aberrations in which lead to numerous degenerative disorders. While the cytoplasmic components of autophagy have been extensively studied, the epigenetic regulation of autophagy genes, especially in stem cells, is less understood. Deciphering the epigenetic regulation of autophagy genes becomes increasingly relevant given the therapeutic benefits of small-molecule epigenetic inhibitors in novel treatment modalities. We observe that, during retinoic acid-mediated differentiation of mouse embryonic stem cells (mESCs), autophagy is induced, and identify the Polycomb group histone methyl transferase EZH2 as a regulator of this process. In mESCs, EZH2 represses several autophagy genes, including the autophagy regulator DNA damage-regulated autophagy modulator protein 1 (Dram1). EZH2 facilitates the formation of a bivalent chromatin domain at the Dram1 promoter, allowing gene expression and autophagy induction during differentiation while retaining the repressive H3K27me3 mark. EZH2 inhibition leads to loss of the bivalent domain, with consequent 'hyper-expression' of Dram1, accompanied by extensive cell death. This study shows that Polycomb group proteins help maintain a balance between autophagy and cell death during stem cell differentiation, in part, by regulating the expression of the Dram1 gene.

Metal mixtures modeling identifies birth weight-associated gene networks in the placentas of children born extremely preterm

Prenatal exposure to toxic metals is linked to numerous adverse birth and later-in-life outcomes. These outcomes are tied to disrupted biological processes in fetal-derived tissues including the placenta and umbilical cord yet the precise pathways are understudied in these target tissues. We set out to examine the relationship between metal concentrations in umbilical cord and altered gene expression networks in placental tissue. These novel relationships were investigated in a subset of the Extremely Low Gestational Age Newborn (ELGAN) cohort (n = 226). Prenatal exposure to 11 metals/metalloids was measured using inductively coupled plasma tandem-mass spectrometry (ICP-MS/MS) in cord tissue, ensuring passage through the placental barrier. RNA-sequencing was used to quantify >37,000 mRNA transcripts. Differentially expressed genes (DEGs) were identified with respect to each metal. Weighted gene co-expression analysis identified gene networks modulated by metals. Two innovative mixtures modeling techniques, namely principal components analysis and quantile-based g-computation, were employed to identify genes/gene networks associated with multi-metal exposure. Individually, lead was associated with the strongest genomic response of 191 DEGs. Joint lead and cadmium exposure was related to 657 DEGs, including DNA Methyl Transferase 1 (DNMT1). These genes were enriched for the Eukaryotic Initiation Factor 2 (EIF2) pathway. Four gene networks, each containing genes within a Nuclear Factor kappa-light-chain-enhancer of Activated B Cells (NF-kB)-mediated network, were significantly increased in average expression level in relation to increases in all metal concentrations. All four of these metal mixture-associated gene networks were negatively correlated with important predictors of neonatal health including birth weight, placenta weight, and fetal growth. Bringing together novel methodologies from epidemiological mixtures analyses and toxicogenomics, applied to a unique cohort of extremely preterm children, the present study highlighted critical genes and pathways in the placenta dysregulated by prenatal metal mixtures. These represent potential mechanisms underlying the developmental origins of metal-induced disease.

Polymethyl methacrylate resin containing ε-poly-L-lysine and 2-methacryloyloxyethyl phosphorylcholine with antimicrobial effects

Statement of problemPolymethyl methacrylate (PMMA) is commonly used in dentistry, including as a denture base material. However, the colonization of a PMMA surface by microbial microorganisms could increase the risk of oral diseases such as denture stomatitis and gingivitis. The development of PMMA with antibacterial properties should improve its clinical application, but whether adding ε-poly-L-lysine (ε-PL) and 2-methacryloyloxyethyl phosphorylcholine (MPC) provides antimicrobial effects is unclear. PurposeThis in vitro study aimed to develop a novel antibacterial PMMA resin containing the natural nontoxic antibacterial agent ε-PL and the protein repellent agent MPC. The mechanical properties, protein repellency, and antimicrobial activities of the resin were then evaluated. Material and methodsDifferent mass fractions of ε-PL and MPC were mixed into PMMA as the experimental groups, with unaltered PMMA as the control group. The flexural strength (n=10) and surface roughness (n=6) of the resulting mixtures were measured to determine their mechanical properties. The antiprotein properties were measured by using the micro bicinchoninic acid method (n=6). The antimicrobial effect of the resin was assessed using live/dead staining (n=6) and methyltransferase (MTT) assays (n=10). According to the variance homogeneity and normal distribution results, 1-way analysis of variance followed by the Tukey honestly significant difference test or the Welch test and the Games-Howell test were used (α=.05 for all tests). ResultsNo significant differences were found in the flexural strength values and surface roughness of the specimens containing 1.5% MPC and 1.5% ε-PL compared with those of the control (P>.05). The addition of ε-PL to the PMMA resin alone significantly increased its bactericidal properties (P<.05). Adding both ε-PL and MPC further increased the antibacterial activity of the PMMA resin without increasing protein adhesion more than in the control group. ConclusionsThe incorporation of both ε-PL and MPC into PMMA improved its antibacterial capacity without affecting its mechanical properties and did not increase protein adhesion. Therefore, the novel PMMA fabricated in this study shows promise for dental applications.

Abstract A032: MUC1-C regulates chromatin remodeling and drives the CSC state in human cancers

Abstract The oncogenic MUC1-C protein evolved in mammals to protect epithelial barrier tissues from the external environment. Chronic activation of MUC1-C promotes lineage plasticity, epigenetic reprogramming, and carcinogenesis. MUC1-C also induces the Yamanaka Pluripotency factors. Chromatin remodeling is critical for the cancer stem cell (CSC) state. Our studies have demonstrated that MUC1-C regulates chromatin remodeling by (i) activating the EZH2/PRC2 complex leading to H3K27 trimethylation, and (ii) the BMI1/PRC1 complex with H2A ubiquitylation. In addition, MUC1-C regulates global changes in chromatin accessibility that are mediated at least in part by JUN/AP-1 and ARID1A/BAF in association with driving the CSC state. We also found that silencing MUC1-C suppresses bulk H3K4 trimethylation and H3K27 acetylation levels in diverse human cancer cells. The COMPASS family of H3K4 methyl transferases (HMT) methylate histone H3K4 and the histone acetyl transferase (HAT) p300/CBP family catalyzes H3K27 acetylation. Involvement of MUC1-C in the regulation of COMPASS has not been previously reported. This study demonstrates that MUC1-C induces COMPASS SET1A and WDR5 gene expression by NF-kB p65-mediated transactivation. WDR5 is required for SET1A complex HMT activity. We found that MUC1-C and WDR5 are necessary for expression of FOS, ATF3 and other AP-1 family members. In a positive feedback loop, MUC1-C, WDR5 and AP-1 were shown to be responsible for induction of genes encoding effectors, including TRAF1 and RELB, in the chronic NF-kB inflammatory response. We also found that MUC1-C, NF-kB, WDR5 and AP-1 are necessary for induction of the (i) KLF4 master regulator of the pluripotency network, and (ii) NOTCH1 effector of stemness. Mechanistically, MUC1-C/NF-kB complexes recruit SET1A/WDR5 and AP-1 to enhancer-like signatures in these target genes, increase the deposition of H3K4me3 with the opening of chromatin, and activate their transcription. These findings demonstrate that MUC1-C activates the COMPASS SET1A/WDR5 complex in integrating the induction of genes that promote chronic inflammation, pluripotency, and the CSC state. Citation Format: trayee Bhattacharya, Atsushi Fushimi, Nami Yamashita, Tao Liu, Song Liu, Donald W. Kufe. MUC1-C regulates chromatin remodeling and drives the CSC state in human cancers. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A032.

Genetics tools for corpora allata specific gene expression in Aedes aegypti mosquitoes

Juvenile hormone (JH) is synthesized by the corpora allata (CA) and controls development and reproduction in insects. Therefore, achieving tissue-specific expression of transgenes in the CA would be beneficial for mosquito research and control. Different CA promoters have been used to drive transgene expression in Drosophila, but mosquito CA-specific promoters have not been identified. Using the CRISPR/Cas9 system, we integrated transgenes encoding the reporter green fluorescent protein (GFP) close to the transcription start site of juvenile hormone acid methyl transferase (JHAMT), a locus encoding a JH biosynthetic enzyme, specifically and highly expressed in the CA of Aedes aegypti mosquitoes. Transgenic individuals showed specific GFP expression in the CA but failed to reproduce the full pattern of jhamt spatiotemporal expression. In addition, we created GeneSwitch driver and responder mosquito lines expressing an inducible fluorescent marker, enabling the temporal regulation of the transgene via the presence or absence of an inducer drug. The use of the GeneSwitch system has not previously been reported in mosquitoes and provides a new inducible binary system that can control transgene expression in Aedes aegypti.

Determination of Metabolomics Profiling in BPA-Induced Impaired Metabolism.

Exposure to bisphenol A (BPA) is unavoidable and it has far-reaching negative effects on living systems. This study aimed to explore the toxic effects of BPA in an experimental animal model through a metabolomics approach that is useful in measuring small molecule perturbations. Beside this, we also examined the ameliorative effects of resveratrol (RSV) against BPA-induced disturbances in experimental mice. This study was conducted for 28 days, and the results showed that BPA indeed induced an impairment in amino acid metabolism, taking place in the mitochondria by significantly (p &lt; 0.05) decreasing the levels of certain amino acids, i.e., taurine, threonine, asparagine, leucine, norleucine, and glutamic acid in the mice plasma. However, the administration of RSV did prove effective against the BPA-induced intoxication and significantly (p &lt; 0.05) restored the level of free amino acids. Lipid metabolites, L-carnitine, sphinganine, phytosphingosine, and lysophosphatidylcholine were also determined in the mice serum. A significant (p &lt; 0.05) decline in glutathione peroxidase (GPx), superoxide dismutase (SOD,) glutathione, and catalase levels and an elevation in malondialdehyde level in the BPA group confirmed the generation of oxidative stress and lipid peroxidation in experimental mice exposed to BPA. The expression of Carnitine palmitoyltransferase I (CPT-I), carnitine palmitoyltransferase II (CPT-II), lecithin-cholesterol acyltransferase (LCAT), carnitine O-octanoyltransferase (CROT), carnitine-acylcarnitine translocase (CACT), and 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR) genes was significantly upregulated in the liver tissue homogenates of experimental mice exposed to BPA, although RSV regulated the expression of these genes when compared with BPA treated experimental mice. CPT-I, CPT-II, and CACT genes are located in the mitochondria and are involved in the metabolism and transportation of carnitine. Hence, this study confirms that BPA exposure induced oxidative stress, upregulated gene expression, and impaired lipid and amino acid metabolism in experimental mice.

A Multicenter, Phase III Study of Chidamide, Azacitidine Combined with CHOP Versus CHOP in Patients with Untreated Peripheral T-Cell Lymphoma

Introduction The biology of peripheral T-cell lymphoma (PTCL) is characterized by recurrent mutations affecting epigenetic regulators such as TET2, DNMT3A, IDH2, and RHOA, particularly in patients with angioimmunoblastic T-cell lymphoma (AITL) or PTCL-not otherwise specified. Azacitidine, a DNA methyl transferase (DNMT) inhibitor, has shown clinical activity as a single agent and in combination in R/R PTCL. Single-agent of the histone deacetylase (HDAC) inhibitors, including romidepsin, belinostat, and chidamide are associated with overall response rates of 25-30% in R/R PTCL. These evidence suggests that PTCL may have a unique vulnerability to epigenetic modulators which also provide the rationale for combining an HDAC inhibitor with a DNMT inhibitor in the backbone of the CHOP regimen forming a novel double-epigenetic regulating chemotherapy as an initial treatment for PTCL. Herein, we report the finding of the phase III trial of chidamide and azacitidine combined with CHOP (AC-CHOP) versus CHOP in patients with untreated PTCL (ClinicalTrials.gov - NCT05075460). Methods This study is a multicenter, non-randomized, phase III trial conducted at 4 centers in China. Eligible patients were aged between 18-75 years with newly diagnosed PTCL. Patients were non-randomly assigned to the AC-CHOP or the CHOP group according to the investigator's and patient's choice. Patients in the AC-CHOP group received azacitidine 200 mg subcutaneously on days 1-2 and 100 mg on day 3, chidamide 20mg orally twice weekly, combined with the CHOP regimen in a 3-week cycle for six cycles. Patients in the CHOP group received the CHOP regimen alone. The primary endpoint was the overall response rate (ORR) after the protocol treatment. Results Between August 2021 and March 2022, 35 patients were enrolled in the study and finished protocol treatment (20 in the AC-CHOP arm and 15 in the CHOP arm). Main pathological subgroups were PTCL not specified (n = 14) and AITL (n = 21). The baseline characteristics and pathological subtypes were mostly similar between the two treatment arms. At the end of treatment, the ORR and complete response (CR) rates were 60.0% and 55.0% in the AC-CHOP arm versus 53.3% and 26.7% in the CHOP arm (P = 0.693 for ORR and P = 0.184 for CR rate) , respectively. With a median follow-up time of 9 months, the median progression-free survival was 8 months in the AC-CHOP arm and 7 months in the CHOP arm (P = 0.602). The most frequent grade 3 to 4 adverse events included neutropenia (55.0% in the AC-CHOP arm versus 40.0% in the CHOP arm), thrombocytopenia (20.0% in the AC-CHOP arm versus 13.3% in the CHOP arm), and Anemia (5.0% in the AC-CHOP arm versus 6.7% in the CHOP arm) which showed no significant difference between the two arms. Conclusions According to the preliminary result of the study, the addition of azacytidine and chidamide to the CHOP regimen did not show a significant improvement of ORR or PFS for patients with untreated PTCL. A trend of higher CR rate was observed which did not reach statistical significance. The safety profile of AC-CHOP regimen was manageable.

PRMT5 Inhibition Enhances Elimination of FLT3-ITD AML Stem Cells in Combination with TKI Treatment

Introduction: The FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) is a commonly observed molecular abnormality in AML and is associated with increased risk of relapse and poor survival. FLT3 tyrosine kinase inhibitors (TKIs) have shown benefit in the treatment of FLT3-ITD AML but do not eliminate primitive leukemia stem cells and are limited by frequent relapse and resistance. There is considerable interest in understanding mechanisms of FLT3-ITD AML stem cell persistence to develop strategies to enhance their elimination. Here we evaluated the role of epigenetic regulators in maintenance of human FLT3-ITD AML cells. Methods: We performed an epigenetic screen using a well-curated collection of epigenetic probes (Epigenetics Probes Collection, Structural Genomics Consortium, n=41). FLT3-ITD (MOLM-13 and MV4-11), and FLT3-WT AML cell lines (OCI-AML3) were exposed to inhibitors for 7 days and CellTiter-Glo and Annexin V labeling were used to evaluate response. We further studied the effects of the PRMT5 inhibitors, GSK-591and LLY-283, with and without FLT3 TKIs, Quizartinib and Giltertinib, on FLT3-ITD+ and FLT3-WT AML cell lines. Synergy between PRMT5 inhibitors and TKIs was analyzed with Calcusyn software using the median effects method of Chou and Talalay. We performed in vivo analysis of the effects of PRMT5 and FLT3 kinase inhibition in a FLT3-ITD Tet2 deleted genetic mouse model of FLT3-ITD AML. Finally, we studied in vitro and in vivo effects of PRMT5 and FLT3 inhibition on primary CD34+ cells from FLT3-ITD AML patients. Results: Screening with epigenetic probes identified PRMT5 arginine methyl transferase inhibitors, GSK591 and LLY-283, amongst the probes with the greatest inhibitory effects on growth and survival of FLT3-ITD MOLM-13 and MV4-11 AML cells. In contrast, inhibitors of PRMT1, PRMT3. PRMT4 and PRMT7 were associated with significantly less growth inhibition of FLT3-ITD AML cell lines. FLT3-WT OCI-AML3 AML cells were also sensitive to PRMT5 inhibition. However, ectopic FLT3-ITD expression in FLT3-WT THP1 cells led to enhanced sensitivity to PRMT5 inhibition. The combination of GSK-591 or LLY-283 with either of the FLT3 TKIs Quizartinib or Giltertinib resulted in synergistically enhanced inhibition of FLT3-ITD+ MOLM-13 and MV4-11 AML cells, compared to TKI or PRMT5 inhibitor alone. On the other hand, FLT3-WT cells were insensitive to TKI alone, and the combination of PRMT5 inhibitor and TKI did not lead to synergistic enhancement of inhibition. We next evaluated the effect of PRMT5 inhibition in a genetic mouse model of FLT3-ITD AML. AML mice were treated with vehicle (control), Giltertinib, LLY283 or the combination of Giltertinib and LLY283 for 2 weeks. AML cells from LLY-283 treated mice showed significantly reduced symmetric dimethylarginine in SmB protein. Significant inhibition of mature AML cells and of AML stem and progenitor cells was seen in the bone marrow and spleen of mice treated with LLY283, and significantly enhanced inhibition was seen with combination of LLY283 and Giltertinib (Figure 1a). Finally, we evaluated the effect of TKI (Giltertinib or Quizartinib), LLY283 or the combination on primary FLT3-ITD AML CD34+ cells with diverse co-mutations (n=8). LLY283 significantly inhibited FLT3-ITD AML CD34+ cell growth and survival in vitro by itself, and the combination of TKI and LLY283 resulted in significantly increased inhibition compared to TKI alone, including both CD34+CD38- and CD34+CD38+ cell populations (Figure 1b). In addition, in vivo treatment of immunodeficient NRGS mice xenografted with human FLT3-ITD AML cells with LLY283 or the combination of Giltertinib and LLY283 led to significant depletion of AML cells in BM and spleen. Conclusion: An epigenetic probe screen identified PRMT5 as a key regulator of FLT3-ITD AML cell viability. We show an important role for PRMT5 in maintenance of murine and human FLT3-ITD AML stem cells and in their persistence following FLT3 TKI treatment. Treatment with a PRMT5 inhibitor in combination with a FLT3 TKI could be a promising approach to enhance elimination of FLT3-ITD AML stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Proximally Biased V(D)J Recombination and Evolution of Non-Productive Clones in B-Cell Precursor Acute Lymphocytic Leukemia with KMT2A::AFF1 Fusion Genes

Background: B-cell precursor acute lymphocytic leukemia (BCP-ALL) carrying KMT2A::AFF1 rearrangements is an aggressive disease with unsatisfactory response to treatment and poor prognosis. KMT2A is a histone methyl transferase involved in the establishment of epigenetic memory. In the fusion protein encoded by the KMT2A::AFF1 fusion gene, the catalytic SET domain of KMT2A mediating H3K4 trimethylation is lost. Instead, the fusion protein interacts with DOT1L and mediates H3K79 methylation. The subversion of the catalytic activity of KMT2A arrests affected cells in an immature stage of development that may precede V(D)J recombination. Aim: Investigate the immune repertoire of KMT2A::AFF1 rearranged BCP-ALL and evaluate the feasibility of measurable residual disease (MRD) detection using immunoglobulin (Ig) gene rearrangements. Patients and Methods: The cohort includes 290 adult and 89 pediatric BCP-ALL patients. The diagnosis was established following WHO classification. WTS (5x107 reads, 2x101bp) data were generated on HiSeq and NovaSeq instruments. Ig gene rearrangements were analyzed using MiXCR and NCBI-IgBlast. Results: We analyzed Ig heavy-chain variable region (IGHV) gene rearrangements in BCP-ALL samples. We observed that the immune repertoire of leukemic cells carrying KMT2A::AFF1 is less diverse than that of other BCP-ALL samples. We investigated the expression levels of IGHV genes using WTS data. We found that IGHV genes located proximally to the IGHD/IGHJ cluster of genes on chr14 were more likely to be expressed in samples carrying KMT2A::AFF1 (Fig 1A). This difference was particularly significant (p<10-18) for IGHV6-1, the IGHV gene closest to the IGHD/IGHJ cluster (Fig 1B). Proximally biased V(D)J recombination is indicative of incomplete Ig locus contraction. Locus contraction is regulated by PAX5, which suppresses WAPL, a negative regulator of chromatin loop extrusion. Chromatin looping of IGHV genes ensures a balanced immune repertoire by bringing distant IGHV genes in proximity to the V(D)J recombination center. We found that in samples with KMT2A::AFF1 fusion genes WAPL expression levels are significantly (p<10-10) higher than in other subtypes of BCP-ALL. Analysis of the Ig gene rearrangements in KMT2A::AFF1 samples revealed abundant expression of alleles encoding non-productive V(D)J rearrangements or IGHV alleles in germline configuration. Only 35% of complementarity determining region 3 covering reads encoded productive rearrangements in KMT2A::AFF1 samples while in other types of BCP-ALL 67% of reads are productive (p<10-6). In samples with chronic lymphocytic leukemia, a tumor of post-germinal center B cells, this percentage is generally 90% or more. Among the non-productive V(D)J rearranged alleles, in-frame stop codons were equally overrepresented as out-of-frame V-J fusions when compared to their abundance in other types of BCP-ALL (p<10-15 in both cases). As light chain rearrangements require signaling from a productive heavy chain, the significantly (p<9x10-7) lower expression of light chain alleles detectable in KMT2A::AFF1 samples supports the notion that the abundant expression of non-productive alleles is not an artefact. Clonal expansion as measured by the Gini index for the number of reads per clonotype is distributed uniformly between 0 (polyclonality) and 1 (monoclonality). Monoclonality was observed in only 10% of cases. Furthermore, some DJ-rearrangements with identical non-templated linker sequences were found to be recombined with different IGHV genes, indicating ongoing V(D)J recombination and clonal evolution. Conclusions: Our data suggest that the KMT2A::AFF1 rearrangement locks the cell of origin at a point of ongoing V(D)J recombination. V(D)J recombination appears to be induced prematurely in these cells. Poor locus contraction could explain the proximally biased V(D)J recombination pattern. The non-sense mediated decay pathway involved in down regulating the expression of non-productive alleles seems to be poorly functional. Consequently, the cells express unusually high amounts of non-productive alleles or alleles in germline configuration that are clonally evolving and derived frequently from IGHV6-1. These features distinguish KMT2A::AFF1 BCP-ALL from other BCP-ALL subtypes and make MRD detection in KMT2A::AFF1 BCP-ALL based on specific Ig gene rearrangements highly error prone. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

RBIO-03. A MULTI-INSTITUTIONAL EXPERIENCE OF GAMMATILE BRACHYTHERAPY AS TREATMENT FOR RECURRENT GLIOBLASTOMAS

Abstract BACKGROUND Recurrent glioblastoma remains a disease without established standard of care. Here, we report the safety profile and survival outcome of recurrent glioblastoma patients treated with maximal safe resection followed by intraoperative placement of GammaTile (GT), a collagen tile-embedded Cs-131 brachytherapy platform. METHODS The study included isocitrate Dehydrogenase (IDH) wild-type glioblastoma patients who suffered tissue confirmed recurrence after concurrent temozolomide-radiation therapy. The series included consecutively patients treated at the University of Minnesota, Barrow Neurological Institute, Vidant Health, and Emory University Hospital. Survival results were stratified by methyl-guanine methyl transferase (MGMT) promoter methylation status RESULTS In total 48 patients were treated between 2019 and 2022 in this multi-institutional experience. The median days of hospitalization following the procedure was two (range: 1-15), with 72.5% of the GT implanted patients discharged home before postoperative day three. In terms of post-operative course, two patients suffered new-onset seizure post-surgery (4.0%). There was one (2%) 30-day mortality from intracranial hemorrhage secondary to heparinization for an ischemic limb. There were 13% readmission within 30 days, including hydrocephalus (n=2), urinary tract infection (n=1), deep venous thrombosis (n=1), failure to thrive (n=1), and infected wound (n=1). Of note, the patient with the infected wound had suffered a previous wound infection prior to the GT implantation and the pathogenic organism for both infections were the same (S. aureus). With a median follow-up of 250 days, twelve-month local control was 82%. Median overall survival (OS) from the time of diagnosis was 19 and 33 months for the MGMT unmethylated and methylated tumors, respectively. Select patients (n = 6) treated with adjuvant ketogenic diet in addition to GT and subsequent chemotherapy exhibited survival beyond expectation (&amp;gt; 14 mo, median survival not reached). CONCLUSION This multi-institutional experience supports further investigation of GT brachytherapy as a treatment for recurrent glioblastomas.

Effects of a New Natural Catechol-O-methyl Transferase Inhibitor on Two In Vivo Models of Parkinson's Disease.

A tetrahydroisoquinoline identified in Mucuna pruriens ((1R,3S)-6,7-dihydroxy-1-methyl-1,2,3,4-tetrahydroisoquinoline-1,3-dicarboxylic acid, compound 4) was synthesized and assessed for its in vitro pharmacological profile and in vivo effects in two animal models of Parkinson's disease. Compound 4 inhibits catechol-O-methyltransferase (COMT) with no affinity for the dopaminergic receptors or the dopamine transporter. It restores dopamine-mediated motor behavior when it is co-administered with L-DOPA to C. elegans worms with 1-methyl-4-phenylpyridinium-damaged dopaminergic neurons. In a 6-hydroxydopamine rat model of Parkinson's disease, its co-administration at 30 mg/kg with L-DOPA enhances the effect of L-DOPA with an intensity similar to that of tolcapone 1 at 30 mg/kg but for a shorter duration. The effect is not dose-dependent. Compound 4 seems not to cross the blood-brain barrier and thus acts as a peripheral COMT inhibitor. COMT inhibition by compound 4 further validates the traditional use of M. pruriens for the treatment of Parkinson's disease, and compound 4 can thus be considered as a promising drug candidate for the development of safe, peripheral COMT inhibitors.

Two-enzyme cascade catalyzed trideuteromethylative modification of natural products

Site-selective incorporation of the trideuterated “magic methyl” group may provide profound pharmacological benefits, which has been considered as an important tool for drug optimization and development in medicinal chemistry. S-adenosyl-L-methionine (SAM) usually acts as the methyl donor under the catalysis of SAM-dependent methyltransferases (MTs), which transfer a methyl group from SAM to substrates, and are widely reported for methylation in natural product biosynthesis. However, it is difficult to introduce other high-value functional groups to substrates in this way since SAM analogs are not easy to obtain in biological methods. Herein we report that trideuterated SAM can be obtained from S-adenosylhomocysterine (SAH) and trideuterated methyl iodide (CD3I) under the participation of halide methyltransferase (HMT). Subsequent one-pot cascades with different MTs achieved the regioselective trideuteromethylation of three natural products, which indicated that trideuterated SAM can be accepted as a co-substrate by C-, N-, O-specific MTs for enzyme-catalyzed trideuteromethylation of natural products.

OR09-2 The RCAN 1.4 Metastasis Progression Suppressor Gene is Hypermethylated at Intron 1 and Downregulated in Papillary Thyroid Cancer

Abstract Progressive metastasis is the proximate cause of cancer-related mortality for the majority of patients with solid tumors. Identifying both drivers, and gatekeepers of late stage progression (metastasis progression suppressors; MPS) is crucial to improve biomarkers and treatments. We previously identified Regulator of Calcineurin 1.4 (RCAN1.4) as a MPS gene in thyroid cancer that functions in part through overexpression of the cap-n-collar transcription factor NFE2L3 (NRF3). However, the mechanisms for RCAN1.4 loss in thyroid cancer, and whether or not this is reversible, had not be addressed. Because the RCAN 1.4 promoter contains CG rich regions reported to be hypermethyalated in other tissues, we hypothesized that RCAN 1.4 downregulation in thyroid cancer was similarly regulated. Studies were performed in both cell lines and de-identified human papillary thyroid cancer (PTC) tumor and normal tissue pairs from our Endocrine Neoplasia Repository tumor bank. Five thyroid cancer cell lines (TPC1, FTC133, BCPAP, C643, and 8505C) with different genetic drivers were selected based on low expression of RCAN 1.4. To determine if lines display methylation-regulated expression of RCAN 1.4, cells were treated with 1-10uM decitabine, a DNA methyl transferase inhibitor. In all cell lines, RCAN1.4 mRNA and protein levels increased with decitabine treatment, consistent with hypermethylation. Analysis of the literature and in silico analysis of the RCAN 1.4 gene identified CG rich regions as putative methylation targets in the proximal promoter and in intron 1. Using two different methods; 1) isolation of MseI-digestion DNA fragments using CpG MethylQuest beads followed by qPCR and 2) methylation-specific PCR (MSP) of bisulfite-modified DNA, hypermethylation of the intron 1 CG rich region was identified. There was no evidence of hypermethylation of the CG rich region of the proximal promoter. Methylation of the two regions also was assessed on 18 pairs PTC and adjacent normal tissues. Similar to the cell lines, hypermethylation was identified at intron 1 of RCAN 1.4 in PTC versus normal tissues. QRT-PCR results confirmed lower levels of RCAN1-4 mRNA in the PTC vs normal tissue. These samples also demonstrated increased NRF3 expression versus normal tissue. Taken together, these data demonstrate that RCAN 1.4 is downregulated in thyroid cancer cells and human PTCs by hypermethylation of CG rich regions in intron 1 and that this effect is reversible using demethylating agents. Presentation: Saturday, June 11, 2022 11:45 a.m. - 12:00 p.m.

A Novel Epigenetic Marker Predicts Treatment Response in Glioblastoma and Provides a Target for Overcoming Treatment Resistance

<h3>Purpose/Objective(s)</h3> Glioblastoma (GBM) is a highly aggressive primary brain tumor. A major challenge in GBM treatment is tumor resistance to radiation and chemotherapy. An important hallmark of GBM is the frequent mutation of epigenetic modifiers resulting in alteration of epigenetic signaling pathways in tumor cells. However, the effect of epigenetic signaling on chemotherapy response in GBM remains to be elucidated. SETD2 is a unique histone methyl transferase that facilitates H3K36 tri-methylation. Here, we unveil the role of SETD2 mutations that frequently occur in GBM in tumor resistance to temozolomide chemotherapy. <h3>Materials/Methods</h3> We have performed next-generation sequencing of the SETD2 gene and immunohistochemical staining if its product H3K36me3 in 129 GBM patient samples that was supplemented with TCGA database analysis of additional 279 GBM samples. We used primary patient-derived GBM lines to investigate the mechanism of chemotherapy resistance in SETD2-mutant GBM. <h3>Results</h3> We demonstrate that inactivating SETD2 mutations occur in up to 13% of GBM patients. Our investigation reveals that SETD2 mutations are associated with reduced progression-free (Median PFS 0.93 vs. 0.44 years, p=0.0022) and overall survival (Median survival 1.69 vs. 1.30 years, p=0.0044) in patients with methylated MGMT (methyl-guanine methyl transferase) promotor who received temozolomide suggesting that mutation of SETD2 is a predictive marker for treatment outcomes in GBM. Consequently, we demonstrate that loss of SETD2 results in reduced H3K36me3 levels in patient samples and a profound resistance to temozolomide in primary patient-derived GBM lines. MGMT-deficient tumors can acquire chemoresistance due to disrupted mismatch repair (MMR), a DNA repair pathway that plays a critical role in converting primary temozolomide-induced DNA lesions into toxic DNA double-strand breaks. Strikingly, we found that SETD2 loss abrogates the expression of the MMR factor MSH6 indicating that chemoresistance in SETD2-deficient cells us due to disrupted MMR. Mechanistically, we show that SETD2 regulates MMR by promoting transcription of the <i>MSH6</i> gene in GBM. Epigenetic modifiers have specific antagonists capable of reversing chromatin alterations induced by these modifiers. This provides a unique opportunity to restore chemotherapy response in SETD2-mutant GBM by targeting the antagonists of SETD2. We demonstrate that combined targeting of H3K36me3-specific histone de-methylases KDM4A and NO66 restores H3K36me3 levels along with MSH6 expression and sensitivity to temozolomide in SETD2-deficient GBM cells. <h3>Conclusion</h3> Our findings establish SETD2 mutation as a novel molecular marker predictive of treatment response in GBM and provide a framework for a novel approach to overcome chemotherapy resistance in this malignant brain tumor by targeting an epigenetic signaling pathway.

EZH2: Its regulation and roles in immune disturbance of SLE.

The pathogenesis of systemic lupus erythematosus (SLE) is related to immune homeostasis imbalance. Epigenetic mechanisms have played a significant role in breaking immune tolerance. Enhancer of zeste homolog 2 (EZH2), the specific methylation transferase of lysine at position 27 of histone 3, is currently found to participate in the pathogenesis of SLE through affecting multiple components of the immune system. This review mainly expounds the mechanisms underlying EZH2-mediated disruption of immune homeostasis in SLE patients, hoping to provide new ideas in the pathogenesis of SLE and new targets for future treatment.

Validation of MRI-Based Models to Predict MGMT Promoter Methylation in Gliomas: BraTS 2021 Radiogenomics Challenge.

Simple SummaryO6-methylguanine-DNA methyl transferase (MGMT) methylation in glioblastoma is an important prognostic and predictive factor that requires an invasive surgical procedure for identification. In several recent studies, MGMT methylation prediction models were developed using MR images, and good diagnostic performance was achieved, which seems to indicate a promising future for radiogenomics. However, the diagnostic performance was not reproducible for numerous research teams when using a larger dataset in the RSNA-MICCAI Brain Tumor Radiogenomic Classification 2021 challenge. To our knowledge, there has been no study regarding the external validation of MGMT prediction models using large-scale multicenter datasets. We tested recent CNN architectures via extensive experiments to investigate whether MGMT methylation in gliomas can be predicted using MRI. With unexpected negative results, approximately 80% of the developed models showed no significant difference with the chance level of 50% in terms of external validation accuracy. In conclusion, MGMT methylation status of gliomas may not be predictable with preoperative MRI, even using deep learning.O6-methylguanine-DNA methyl transferase (MGMT) methylation prediction models were developed using only small datasets without proper external validation and achieved good diagnostic performance, which seems to indicate a promising future for radiogenomics. However, the diagnostic performance was not reproducible for numerous research teams when using a larger dataset in the RSNA-MICCAI Brain Tumor Radiogenomic Classification 2021 challenge. To our knowledge, there has been no study regarding the external validation of MGMT prediction models using large-scale multicenter datasets. We tested recent CNN architectures via extensive experiments to investigate whether MGMT methylation in gliomas can be predicted using MR images. Specifically, prediction models were developed and validated with different training datasets: (1) the merged (SNUH + BraTS) (n = 985); (2) SNUH (n = 400); and (3) BraTS datasets (n = 585). A total of 420 training and validation experiments were performed on combinations of datasets, convolutional neural network (CNN) architectures, MRI sequences, and random seed numbers. The first-place solution of the RSNA-MICCAI radiogenomic challenge was also validated using the external test set (SNUH). For model evaluation, the area under the receiver operating characteristic curve (AUROC), accuracy, precision, and recall were obtained. With unexpected negative results, 80.2% (337/420) and 60.0% (252/420) of the 420 developed models showed no significant difference with a chance level of 50% in terms of test accuracy and test AUROC, respectively. The test AUROC and accuracy of the first-place solution of the BraTS 2021 challenge were 56.2% and 54.8%, respectively, as validated on the SNUH dataset. In conclusion, MGMT methylation status of gliomas may not be predictable with preoperative MR images even using deep learning.

The lysine methyltransferases SET and MYND domain containing 2 (Smyd2) and Enhancer of Zeste 2 (Ezh2) co-regulate osteoblast proliferation and mineralization

Bone formation is controlled by histone modifying enzymes that regulate post-translational modifications on nucleosomal histone proteins and control accessibility of transcription factors to gene promoters required for osteogenesis. Enhancer of Zeste homolog 2 (EZH2/Ezh2), a histone H3 lysine 27 (H3K27) methyl transferase, is a suppressor of osteoblast differentiation. Ezh2 is regulated by SET and MYND domain-containing protein 2 (SMYD2/Smyd2), a lysine methyltransferase that modifies both histone and non-histone proteins. Here, we examined whether Smyd2 modulates Ezh2 suppression of osteoblast differentiation. Musculoskeletal RNA-seq data show that SMYD2/Smyd2 is the most highly expressed SMYD/Smyd member in human bone tissues and mouse osteoblasts. Smyd2 loss of function analysis in mouse MC3T3 osteoblasts using siRNA depletion enhances proliferation and calcium deposition. Loss of Smyd2 protein does not affect alkaline phosphatase activity nor does it result in a unified expression response for standard osteoblast-related mRNA markers (e.g., Bglap, Ibsp, Spp1, Sp7), indicating that Smyd2 does not directly control osteoblast differentiation. Smyd2 protein depletion enhances levels of the osteo-suppressive Ezh2 protein and H3K27 trimethylation (H3K27me3), as expected from increased cell proliferation, while elevating the osteo-inductive Runx2 protein. Combined siRNA depletion of both Smyd2 and Ezh2 protein is more effective in promoting calcium deposition when compared to loss of either protein. Collectively, our results indicate that Smyd2 inhibits proliferation and indirectly the subsequent mineral deposition by osteoblasts. Mechanistically, Smyd2 represents a functional epigenetic regulator that operates in parallel to the suppressive effects of Ezh2 and H3K27 trimethylation on osteoblast differentiation.

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Characterization of benzylisoquinoline alkaloid methyltransferases in Liriodendron chinense provides insights into the phylogenic basis of angiosperm alkaloid diversity.

Benzylisoquinoline alkaloids (BIAs) are a class of plant secondary metabolites with great pharmacological value. Their biosynthetic pathways have been extensively elucidated in the species from the Ranunculales order, such as poppy and Coptis japonica, in which methylation events play central roles and are directly responsible for BIA chemodiversity. Here, we combined BIA quantitative profiling and transcriptomic analyses to identify novel BIA methyltransferases (MTs) from Liriodendron chinense, a basal angiosperm plant. We identified an N-methyltransferase (LcNMT1) and two O-methyltransferases (LcOMT1 and LcOMT3), and characterized their biochemical functions in vitro. LcNMT1 methylates (S)-coclaurine to produce mono- and dimethylated products. Mutagenesis experiments revealed that a single-residue alteration is sufficient to change its substrate selectivity. LcOMT1 methylates (S)-norcoclaurine at the C6 site and LcOMT3 methylates (S)-coclaurine at the C7 site, respectively. Two key residues of LcOMT3, A115 and T301, are identified as important contributors to its catalytic activity. Compared with Ranunculales-derived NMTs, Magnoliales-derived NMTs were less abundant and had narrower substrate specificity, indicating that NMT expansion has contributed substantially to BIA chemodiversity in angiosperms, particularly in Ranunculales species. In summary, we not only characterized three novel enzymes that could be useful in the biosynthetic production of valuable BIAs but also shed light on the molecular origin of BIAs during angiosperm evolution.