The DNA methyltransferase inhibitors zebularine and decitabine induce mitochondria-mediated apoptosis and DNA damage in p53 mutant leukemic T cells

ABSTRACTγδ T cells are a distinct subgroup of T cells that bridge the innate and adaptive immune systems and can attack cancer or virus-infected cells in an MHC-unrestricted manner. Despite its antitumor ability in both autologous and allogeneic settings, earlier trials of adoptive γδ T cell transfer in solid tumors had limited success due to limitations in cell expansion and the lack of a strategy to modulate tumor lytic interactions between γδ T and cancer cells. Here, we show through quantitative surface proteomics and gene enrichment analyses that DNA methyltransferase inhibitors (DNMTis) upregulate multiple surface molecules related to γδ T cell activation in cancer cells. DNMTi treatment of human lung cancer potentiates tumor lysis by ex vivo-expanded γδ T cells using a clinical-grade expansion protocol developed by our team to enrich for the Vδ1 subset while preserving their antitumor effector functions. Mechanistically, DNMTis enhance immune synapse formation and stabilize the synaptic cleft to facilitate γδ T-mediated tumor lysis. Through integrated analysis of RNA-seq, DNA methylation, and ATAC-seq, we demonstrate that depletion of DNMTs induces coordinated pattern alterations of immune synaptic-cytoskeletal networks at the cancer side of the immune synapse. In addition, single-cell mass cytometry reveals enrichment of polyfunctional γδ T subsets by DNMTis. Combined DNMTi and adoptive γδ T transfer in a mouse lung cancer model offers a significant survival benefit. Consistently, the DNMTi-associated cytoskeleton signature identifies a subset of lung cancer patients with improved survival. Our results demonstrate that epigenetic mechanisms are crucial for cytoskeletal remodeling in cancer to potentiate immune attack and support a combinatorial strategy of DNMTis and γδ T cell-based immunotherapy in lung cancer management.One Sentence SummaryDNA methyltransferase inhibitors potentiate the killing of lung cancer by γδ T cells through remodeling cytoskeletal-immune synaptic networks.

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DNA methyltransferase inhibitors modulate histone methylation: epigenetic crosstalk between H3K4me3 and DNA methylation during sperm differentiation

Zygote ◽

10.1017/s0967199420000684 ◽

2021 ◽

pp. 1-6

Author(s):

Liliana Burlibaşa ◽

Alina-Teodora Nicu ◽

Carmen Domnariu

Keyword(s):

Dna Methylation ◽

Histone Methylation ◽

Dna Methyltransferase ◽

Integrated Approach ◽

Regulatory Mechanisms ◽

Temporal Expression ◽

Dna Methyltransferase Inhibitors ◽

Expression Of Genes ◽

Species Survival ◽

Advanced Stages

Summary The process of cytodifferentiation in spermatogenesis is governed by a unique genetic and molecular programme. In this context, accurate ‘tuning’ of the regulatory mechanisms involved in germ cells differentiation is required, as any error could have dramatic consequences on species survival and maintenance. To study the processes that govern the spatial–temporal expression of genes, as well as analyse transmission of epigenetic information to descendants, an integrated approach of genetics, biochemistry and cytology data is necessary. As information in the literature on interplay between DNA methylation and histone H3 lysine 4 trimethylation (H3K4me3) in the advanced stages of murine spermatogenesis is still scarce, we investigated the effect of a DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine, at the cytological level using immunocytochemistry methodology. Our results revealed a particular distribution of H3K4me3 during sperm cell differentiation and highlighted an important role for regulation of DNA methylation in controlling histone methylation and chromatin remodelling during spermatogenesis.

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Expanding the Structural Diversity of DNA Methyltransferase Inhibitors

Pharmaceuticals ◽

10.3390/ph14010017 ◽

2020 ◽

Vol 14 (1) ◽

pp. 17

Author(s):

K. Eurídice Juárez-Mercado ◽

Fernando D. Prieto-Martínez ◽

Norberto Sánchez-Cruz ◽

Andrea Peña-Castillo ◽

Diego Prada-Gracia ◽

...

Keyword(s):

Histone Deacetylases ◽

Dna Methyltransferase ◽

Structural Diversity ◽

Dna Methyltransferases ◽

Dna Methyltransferase Inhibitors ◽

Epigenetic Drug ◽

Ic50 Values ◽

Dietary Component ◽

Approved Drugs ◽

Epigenetic Processes

Inhibitors of DNA methyltransferases (DNMTs) are attractive compounds for epigenetic drug discovery. They are also chemical tools to understand the biochemistry of epigenetic processes. Herein, we report five distinct inhibitors of DNMT1 characterized in enzymatic inhibition assays that did not show activity with DNMT3B. It was concluded that the dietary component theaflavin is an inhibitor of DNMT1. Two additional novel inhibitors of DNMT1 are the approved drugs glyburide and panobinostat. The DNMT1 enzymatic inhibitory activity of panobinostat, a known pan inhibitor of histone deacetylases, agrees with experimental reports of its ability to reduce DNMT1 activity in liver cancer cell lines. Molecular docking of the active compounds with DNMT1, and re-scoring with the recently developed extended connectivity interaction features approach, led to an excellent agreement between the experimental IC50 values and docking scores.

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GATA3 induces mitochondrial biogenesis in primary human CD4+ T cells during DNA damage

Nature Communications ◽

10.1038/s41467-021-23715-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lauren A. Callender ◽

Johannes Schroth ◽

Elizabeth C. Carroll ◽

Conor Garrod-Ketchley ◽

Lisa E. L. Romano ◽

...

Keyword(s):

Dna Damage ◽

T Cells ◽

T Cell ◽

Mitochondrial Biogenesis ◽

Cell Manipulation ◽

Th2 Cells ◽

Related Factor ◽

Mitochondrial Mass ◽

T Helper 2 ◽

And Function

AbstractGATA3 is as a lineage-specific transcription factor that drives the differentiation of CD4+ T helper 2 (Th2) cells, but is also involved in a variety of processes such as immune regulation, proliferation and maintenance in other T cell and non-T cell lineages. Here we show a mechanism utilised by CD4+ T cells to increase mitochondrial mass in response to DNA damage through the actions of GATA3 and AMPK. Activated AMPK increases expression of PPARG coactivator 1 alpha (PPARGC1A or PGC1α protein) at the level of transcription and GATA3 at the level of translation, while DNA damage enhances expression of nuclear factor erythroid 2-related factor 2 (NFE2L2 or NRF2). PGC1α, GATA3 and NRF2 complex together with the ATR to promote mitochondrial biogenesis. These findings extend the pleotropic interactions of GATA3 and highlight the potential for GATA3-targeted cell manipulation for intervention in CD4+ T cell viability and function after DNA damage.

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Cytotoxicity, DNA Damage, and Apoptosis Induced by New Fotemustine Analogs on Human Melanoma Cells in Relation to O6-Methylguanine DNA-Methyltransferase Expression

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.103.051938 ◽

2003 ◽

Vol 307 (2) ◽

pp. 816-823 ◽

Cited By ~ 18

Author(s):

Isabelle Passagne ◽

Alexandre Evrard ◽

Jean-Yves Winum ◽

Philippe Depeille ◽

Pierre Cuq ◽

...

Keyword(s):

Dna Damage ◽

Dna Methyltransferase ◽

Melanoma Cells ◽

Human Melanoma ◽

Methylguanine Dna Methyltransferase ◽

Human Melanoma Cells

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EXTH-39. DUAL CHLOROTOXIN CAR AND MGMT ΓΔ T CELLS FOR DRUG RESISTANT IMMUNOTHERAPY OF GLIOBLASTOMA MULTIFORME

Neuro-Oncology ◽

10.1093/neuonc/noab196.678 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi172-vi172

Author(s):

Lei Ding ◽

Lawrence Lamb

Keyword(s):

T Cells ◽

Glioblastoma Multiforme ◽

Dna Methyltransferase ◽

Γδ T Cells ◽

Scorpion Venom ◽

Transduction Efficiency ◽

Potential Candidate ◽

Jurkat T Cells ◽

Binding Domains ◽

Car T

Abstract While recent advances in immunotherapies have shown promise in extracranial tumors, Glioblastoma Multiforme (GBM) has remained challenging. We have previously developed a novel approach to the treatment of primary GBM by combining simultaneous intracranial administration of gene-modified γδ T cells expressing O-6-Methylguanine-DNA Methyltransferase (MGMT) and standard temozolomide (TMZ) chemotherapy. To further improve tumor targeting and the efficacy of the γδ T cell against GBM, we modified γδ T cells with a CAR using chlorotoxin (CLTX), a small peptide derived from scorpion venom that specifically binds the glioma-specific chloride channel, as binding domains (CLTX-CARs). MGMTp140k was co-expressed within the same CLTX-CAR vectors to confer TMZ resistance to the CAR-T cells. We optimized the CAR constructs as CLTX-CD8a-CD28TM-CD28co-CD3z-P2A-MGMT and the activation of CD69 was verified in CLTX-CAR transduced Jurkat T cells with GBM cell co-culture. We further tested dual-CLTX-CAR constructs with 2x CLTX peptide as binding domains (dCLTX-CAR) and demonstrated dCLTX-CAR-Jurkat T cells can activate T cells efficiently with improved persistence compared to the mono-CLTX-CAR-Jurkat T cells 3 weeks after transduction (84% vs 37%). In order to preserve the powerful innate-recognition properties of γδ T cells, we developed CLTX-CARs without CD3z activation domain (NoZ-CLTX-CAR) to mitigate activation induced cell death (AICD) and tonic signaling issues. NoZ-CLTX-CAR-Jurkat T cells showed no CD69 activation with GBM cells co-culture and superior CAR-T persistence compared to CLTX-CARs with a CD3z signaling domain. Furthermore, we were able to efficiently transduce (up to 70% transduction efficiency) γδ T cells with NoZ-dCLTX-CAR lentiviral which demonstrated enhanced cytotoxicity against GBM cells compare to non-transduced control γδ T cells. Our approach of combining the dCLTX-CAR and TMZ resistance will be further validated in animal model experiments and could be a potential candidate for clinical development for GBM.

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RECQL5 mutations as a potential efficacious predictor of immunotherapy in melanoma patients.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.e21547 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. e21547-e21547

Author(s):

Yun Zhao ◽

Dandan Fan ◽

Xiaochun Huang ◽

Mengli Huang ◽

Yaoxu Chen ◽

...

Keyword(s):

Dna Damage ◽

T Cells ◽

Immune Cells ◽

Statistical Significance ◽

Dna Damage Repair ◽

Objective Response ◽

Wilcoxon Test ◽

Durable Response ◽

Damage Repair ◽

Melanoma Patients

e21547 Background: Gene mutations in DNA damage repair (DDR) pathway were reported to affect the clinical response to immune checkpoint inhibitors (ICIs) by driving mutagenicity. Previous studies showed that RECQL5 is involved in RAD51-mediated strand invasion for homologous recombination DNA damage repair (HR-DDR). RECQL5 encoding RecQ protein-like 5 (RECQL5) is a member of RecQ helicase family and was discovered to play a tumor-suppressive or oncogenic role in various cancers. However, the association between RECQL5 mutation and ICI efficacy has not been revealed. Methods: Data of nine publicly independent cohorts of NSCLC, melanoma, and pan-cancer were retrieved (Rizvi, MSKCC, OAK/POPLAR, Van Allen, Hugo, Synder, Miao and Samstein cohorts) to investigate the correlation between RECQL5 mutations and clinical events including overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and clinical benefit rate (CBR). Wilcoxon test was used for comparing tumor mutational burden (TMB) between RECQL5-mutated patients and their wild-type counterparts. In addition, the correlation between infiltration of immune cells and RECQL5 mutation status was also analyzed by QUANTISEO using data of SKCM cohort (n=466) from TCGA database. Statistical significance was set at P = 0.05. Results: RECQL5 mutations were most commonly seen in melanoma patients and the detection rate was 4%-8% in melanoma patients from cohort Van Allen, Hugo, Synder, and Miao. RECQL5 mutation was not detected in other cancers except for one lung cancer case. In the three melanoma cohorts (Van Allen, Synder, Miao) with OS data available, the OS of the RECQL5-mutated patients was 2 to 3 times longer than that of the RECQL5-wt patients. In Synder cohort, the difference in OS between these two subsets was statistically significant (53.9 months vs 25.0 months, P = 0.045). In addition, the occurrence of RECQL5 mutations was correlated with higher CBR both in Miao ( P = 0.017) and Synder cohorts ( P = 0.011). No difference was observed in the PFS or ORR between these two subsets. Notably, RECQL5 mutations were associated with higher TMB levels both in Miao (32.6 mut/Mb vs 7.9 mut/Mb, P = 0.016) and Synder cohorts (1124 mut/Mb vs 358 mut/Mb, P = 0.033). Moreover, RECQL5 mutations were found to be correlated with increased infiltration of CD4+ T cells (no-regulatory) ( P = 0.037), B cells ( P = 0.13) and decreased infiltration of regulatory CD4+ T cells ( P = 0.02) in RECQL5-mutated tumors over RECQL5-wt tumors. Conclusions: RECQL5 mutations can serve as a potential predictor for a durable response to ICIs in melanoma. Moreover, the occurrence of RECQL5 mutations was correlated with higher TMB level as well as infiltration of immune cells, indicating the underlying mechanism of its predictive effect.

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