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Purpose: SETD2 aberrancy, as the main reason of depleted levels of H3K36me3, plays an important role in diverse forms of cancer and is most notably in clear cell renal cell carcinoma (ccRCC). Altered chromatin modelers related to impaired H3K36me3 occupancy are linked to renal cancer aggressiveness and metastasis, where the latter dramatically drops the relative 5-year survival rate of ccRCC from 91% to 11%. Developing novel treatment targeting H3K36me3-compromised cancer cell is urgently needed for diagnostic and treatment purposes. Methods: Phenotypic and animal experiments were conducted to test the sensitivity of kidney cancer cells with impaired H3K36me3 to hypomethylating agent (HMA), PARP inhibitor (PARPi), and HMA + PARPi combination treatments. Comet assay, flow cytometry, western blot and bioinformatic analysis were performed to explore the mechanism beneath. Results: Combination treatment of 5-Aza-CdR (DAC) and Talazoparib (PARPi) generate more cytotoxicity in SETD2-altered ccRCC cell lines than in SETD2-intact cell lines, including inhibited cell growth, incomplete colony formation, more apoptosis, increased DNA damage and impaired DNA damage repair. The up-regulation of STING and transposable element (TE), as well as the loss of chromatin stability, were more remarkable in SETD2-altered ccRCC cell lines when treated with DAC + PARPi. Conclusion: Combination treatment of DAC and Talazoparib showed greater synergistic anti-tumor effect in SETD2 aberrant than in SETD2 wild-type ccRCC cells, making it a potential treatment strategy for H3K36me3-compromised cancers. Further pre-clinical studies should be conducted to make a precise medicine-based approach. Citation Format: Xinyi Zhou, Guanghou Fu, Hong-Tao Li, Hemant Gujar, Daniel J. Weisenberger, David I. Quinn, Bo Han, Gangning Liang. SETD2 aberrancy enhanced the synergetic anti-tumor effects of DNA hypomethylating agents and PARP inhibitors in aggressive clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3736.

Large-scale sequencing efforts of human cancers have identified recurrent mutations and deletions in a variety of chromatin-regulating proteins that modulate DNA methylation, histone modifications, and nucleosome positioning. The dysregulation of histone H3 lysine 36 (H3K36) methyltransferase, SETD2, through transcriptional or genetic aberrations is associated with worse clinical outcomes and metastasis in kidney cancer. Limited targeted therapeutic interventions are available for aggressive SETD2-mutant ccRCC tumors. Here, we reveal that kidney cancer cells displaying diminished H3K36 trimethylation levels, a consequence of SETD2 deficiency, show increased sensitivity of anti-tumor effects to DNA hypomethylating agent (HMA). We discovered that HMA treatment induced stronger viral mimicry activation and immune upregulation, which is potentiated by higher transposable element (TE) expression in SETD2-mutant cancer cells. Mechanistically, we provide evidence that substantial number of the HMA-induced TE expression is a consequence of mis-splicing, which is associated deficient in slicing in SETD2-loss content along with rapid gain of H3K9me3 across exons. These all suggested HMA could turn immune-cold tumor to immune-hot tumor and sensitize tumors to immune therapy. Then we performed in vivo assay in immune competent mice. Indeed, SETD2 deficient tumors were extremely sensitive to combination treatment of HMA and immune checkpoint inhibitor. Our finding provides one of the first preclinical and in vivo evidence that demonstrates the SETD2 dysregulation can be an epigenetic therapeutic target in ccRCC, especially in combination with immune checkpoint inhibitors, for future clinical trials. Citation Format: Hong-Tao Li, Hyo Sik Jang, Krizia Rohena-Rivera, Hemant Gujar, Minmin Liu, Justin Kulchycki, Xinyi Zhou, Shuqing Zhao, Peter Jones, Neil Bhowmick, Gangning Liang. Loss of SETD2 sensitizes kidney cancer cells to DNA methylation inhibitors by inducing viral mimicry and RNA mis-splicing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3618.

Highly tumorigenic, drug-resistant cancer stem-like cells drive cancer progression. These aggressive cells can arise repeatedly from bulk tumor cells independently of mutational events, suggesting an epigenetic mechanism. To test this possibility, we studied bladder cancer cells as they cyclically shifted to and from a cancer stem-like phenotype, and we discovered that these two states exhibit distinct DNA methylation and chromatin accessibility. Most differential chromatin accessibility was independent of methylation and affected the expression of driver genes such as E2F3, a cell cycle regulator associated with aggressive bladder cancer. Cancer stem-like cells exhibited increased E2F3 promoter accessibility and increased E2F3 expression that drove cell migration, invasiveness and drug resistance. Epigenetic interference using a DNA methylation inhibitor blocked the transition to a cancer stem-like state and reduced E2F3 expression. Our findings indicate that epigenetic plasticity plays a key role in the transition to and from an aggressive, drug-resistant phenotype.

Purpose: With the current standard of care for Pediatric high-grade glioma (pHGG) patients the 5-year survival rate remain low at 5-35%. Thus, there is an urgent need to develop improved therapies for pHGG patients. A specific point mutation of histone H3 (H3F3A) has been identified at amino acid 27 (lysine to methionine (K27M)), resulting in inhibition of EZH2 (histone H3K27 tri-methylase), and reduced global levels of H3K27 tri-methylation (H3K27me3). The H3K27M alteration can be found in the majority (~80%) of pHGGs and represents an attractive target for diagnostic and treatment. Since the H3K27me3 mark correlate with gene silencing, mutations resulting in global decrease in H3K27me3 may result in aberrant gene regulation. Majority of demethylated promoters remain silenced after treatment with hypomethylating agent (HMA) due to the presence of repressive H3K27me3. HMAs treatment is also rendered ineffective in case of repression of endogenous retroviruses (ERVs) due to the presence of H3K27me3. Hence, pHGGs harboring the H3K27M mutation with low levels of H3K27me3 represent an attractive therapeutic target for treatment with HMAs. HMAs are approved by FDA for treatment of several forms of human cancers. Methods: We had introduced mutant H3K27M in human pediatric glioma cell (SF188). Pediatric glioma cell lines (wild type and endogenous or endogenous expressed with H3K27M) were treated with DNA hypomethylating agents (HMAs). Cell doubling time was calculated. DNA methylation and gene expression were monitored at different time points. Additional RNA-seq data were obtained from pediatric glioma patient and patient derived cell lines. Results: We demonstrate that pHGG cells carrying H3K27M mutation are more aggressive and showed down-regulation of innate immune response genes (immune deficient or immune cold) and up-regulation of cancer stem like genes as compared to the wild-type pHGG cells. These findings are further confirmed with published data from patient-derived pHGG cells. The prolonged antitumor effects in pHGG cells with H3K27M mutation observed after HMA treatment may be due to the failure of DNA remethylation and upregulated innate immune response pathway genes due to ERV reactivation. Conclusion: Our findings suggest that HMA treatment may be effective against pHGGs with the H3K27M mutation and critical for converting immune deficient cells into immune reactive cells (immune hot). The success of this project will lead to clinical trials for pHGG patients to evaluate HMAs with immune-based therapies in pHGG patients, for whom there are currently no effective treatments. Citation Format: Hemant Gujar, Lian Zhang, Hong-Tao Li, Hitoshi Otani, Daniel J. Weisenberger, Anat Erdreich-Epstein, Gangning Liang. The mutated chromatin modifier gene, H3F3AK27M, is a druggable target of DNA hypomethylating agents in pediatric high-grade glioma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 462.

A DNA sequence is the hard copy of the human genome and it is a driving force in determining the physiological processes in an organism. Concurrently, the chemical modification of the genome and its related histone proteins is dynamically involved in regulating physiological processes and diseases, which overall constitutes the epigenome network. Among the various forms of epigenetic modifications, DNA methylation at the C-5 position of cytosine in the cytosine⁻guanine (CpG) dinucleotide is one of the most well studied epigenetic modifications. DNA methyltransferases (DNMTs) are a family of enzymes involved in generating and maintaining CpG methylation across the genome. In mammalian systems, DNA methylation is performed by DNMT1 and DNMT3s (DNMT3A and 3B). DNMT1 is predominantly involved in the maintenance of DNA methylation during cell division, while DNMT3s are involved in establishing de novo cytosine methylation and maintenance in both embryonic and somatic cells. In general, all DNMTs require accessory proteins, such as ubiquitin-like containing plant homeodomain (PHD) and really interesting new gene (RING) finger domain 1 (UHRF1) or DNMT3-like (DNMT3L), for their biological function. This review mainly focuses on the role of DNMT3B and its isoforms in de novo methylation and maintenance of DNA methylation, especially with respect to their role as an accessory protein.

To begin studies on reproduction in common bed bug, Cimex lectularius, we identified three genes coding for vitellogenin (Vg, a protein required for the reproductive success of insects) and studied their hormonal regulation. RNA interference studied showed that expression of Vg3 gene in the adult females is a prerequisite for successful completion of embryogenesis in the eggs laid by them. Juvenile hormone (JH) receptor, Methoprene-tolerant (Met), steroid receptor coactivator (SRC) and GATAa but not ecdysone receptor (EcR) or its partner, ultraspiracle (USP) are required for expression of Vg genes. Feeding and mating working through Vg, Met, SRC, EcR, and GATAa regulate oocyte development. Knockdown of the expression of Met, SRC, EcR, USP, BR-C (Broad-Complex), TOR (target of rapamycin), and GATAa in female adults resulted in a reduction in the number eggs laid by them. Interestingly, Kruppel homolog 1 (Kr-h1) knockdown in the adult females did not reduce their fecundity but affected the development of embryos in the eggs laid by females injected with Kr-h1 double-stranded RNA. These data suggest that JH functioning through Met and SRC regulate both vitellogenesis and oogenesis in C. lectularius. However, JH does not work through Kr-h1 but may work through transcription factors not yet identified.

The bed bug, Cimex lectularius, has re-established itself as a ubiquitous human ectoparasite throughout much of the world during the past two decades. This global resurgence is likely linked to increased international travel and commerce in addition to widespread insecticide resistance. Analyses of the C. lectularius sequenced genome (650 Mb) and 14,220 predicted protein-coding genes provide a comprehensive representation of genes that are linked to traumatic insemination, a reduced chemosensory repertoire of genes related to obligate hematophagy, host–symbiont interactions, and several mechanisms of insecticide resistance. In addition, we document the presence of multiple putative lateral gene transfer events. Genome sequencing and annotation establish a solid foundation for future research on mechanisms of insecticide resistance, human–bed bug and symbiont–bed bug associations, and unique features of bed bug biology that contribute to the unprecedented success of C. lectularius as a human ectoparasite., The bed bug, Cimex lectularius, is a ubiquitous human ectoparasite with global distribution. Here, the authors sequence the genome of the bed bug and identify reductions in chemosensory genes, expansion of genes associated with blood digestion and genes linked to pesticide resistance.

The common bed bug is an obligate hematophagous parasite of humans. We studied the regulation of molting and metamorphosis in bed bugs with a goal to identify key players involved. qRT-PCR studies on the expression of genes known to be involved in molting and metamorphosis showed high levels of Krüppel homolog 1 [Kr-h1, a transcription factor that plays key roles in juvenile hormone (JH) action] mRNA in the penultimate nymphal stage (N4). However, low levels of Kr-h1 mRNA were detected in the fifth and last nymphal stage (N5). Knockdown of Kr-h1 in N4 resulted in a precocious development of adult structures. Kr-h1 maintains the immature stage by suppressing E93 (early ecdysone response gene) in N4. E93 expression increases during the N5 in the absence of Kr-h1 and promotes the development of adult structures. Knockdown of E93 in N5 results in the formation of supernumerary nymphs. The role of JH in the suppression of adult structures through interaction with Kr-h1 and E93 was also studied by the topical application of JH analog, methoprene, to N5. Methoprene induced Kr-h1 and suppressed E93 and induced formation of the supernumerary nymph. These data show interactions between Kr-h1, E93 and JH in the regulation of metamorphosis in the bed bugs.

Recent advances in genomic and post-genomic technologies have facilitated a genome-wide analysis of the insecticide resistance-associated genes in insects. Through bed bug, Cimex lectularius transcriptome analysis, we identified 14 molecular markers associated with pyrethroid resistance. Our studies revealed that most of the resistance-associated genes functioning in diverse mechanisms are expressed in the epidermal layer of the integument, which could prevent or slow down the toxin from reaching the target sites on nerve cells, where an additional layer of resistance (kdr) is possible. This strategy evolved in bed bugs is based on their unique morphological, physiological and behavioral characteristics and has not been reported in any other insect species. RNA interference-aided knockdown of resistance associated genes showed the relative contribution of each mechanism towards overall resistance development. Understanding the complexity of adaptive strategies employed by bed bugs will help in designing the most effective and sustainable bed bug control methods.