Histone Methyltransferase Activity Research Articles

Histone Methylation-Mediated Reproductive Toxicity to Consumer Product Chemicals in Caenorhabditis elegans: An Epigenetic Adverse Outcome Pathway (AOP).

The significance of histone methylation in epigenetic inheritance underscores its relevance to disease and the chronic effects of environmental chemicals. However, limited evidence of the causal relationships between chemically induced epigenetic changes and organismal-level effects hinders the application of epigenetic markers in ecotoxicological assessments. This study explored the contribution of repressive histone marks to reproductive toxicity induced by chemicals in consumer products in Caenorhabditis elegans, applying the adverse outcome pathway (AOP) framework. Triclosan (TCS) and tetrabromobisphenol A (TBBPA) exposures caused reproductive toxicity and altered histone methyltransferase (HMT) and histone demethylase (HDM) activities, increasing the level of trimethylation of H3K9 and H3K27. Notably, treatment with an H3K27-specific HMT inhibitor alleviated reproductive defects and the transcriptional response of genes related to vitellogenin, xenobiotic metabolism, and oxidative stress. Comparison of points of departure (PODs) based on calculated benchmark concentrations (BMCs) revealed the sensitivity of histone-modifying enzyme activities to these chemicals. Our findings suggest that the 'disturbance of HMT and HDM' can serve as the molecular initiating event (MIE) leading to reproductive toxicity in the epigenetic AOP for TCS and TBBPA. The study extended the biological applicability of these enzymes by identifying model species with analogous protein sequences and functions. This combined approach enhances the essentiality, empirical support, and taxonomic domain of applicability (tDOA), which are crucial considerations for ecotoxicological AOPs. Given the widespread use and environmental distribution of chemicals in consumer products, this study proposes histone-modifying enzyme activity as an effective screening tool for reproductive toxicants and emphasizes the integration of epigenetic mechanisms into a prospective ERA.

WTAP-dependent N6-methyladenosine methylation of lncRNA TEX41 promotes renal cell carcinoma progression.

The methyltransferase Wilms' tumor 1-associated protein (WTAP) has been reported to be dysregulated in various tumors. However, its role in renal cell carcinoma (RCC) remains elusive. Here, we explored whether WTAP was upregulated in RCC specimens compared to normal tissues. Functionally, WTAP promoted RCC cell proliferation and metastasis in vivo and in vitro. Mechanistically, WTAP act as an N6-methyladenosine transferase to regulate the m6A modification of long noncoding RNA TEX41. Then, the upregulated m6A modification destabilized TEX41 in a YTHDF2-dependent manner. Furthermore, TEX41 interacted with the SUZ12 protein and increased the histone methyltransferase activity of SUZ12, resulting in HDAC1 silencing. Totally, our study demonstrated the oncogenic the role of WTAP/TEX41/SUZ12/HDAC1 axis in RCC progression.

PRDM16 determines specification of ventricular cardiomyocytes by suppressing alternative cell fates.

PRDM16 is a transcription factor with histone methyltransferase activity expressed at the earliest stages of cardiac development. Pathogenic mutations in humans lead to cardiomyopathy, conduction abnormalities, and heart failure. PRDM16 is specifically expressed in ventricular but not atrial cardiomyocytes, and its expression declines postnatally. Because in other tissues PRDM16 is best known for its role in binary cell fate decisions, we hypothesized a similar decision-making function in cardiomyocytes. Here, we demonstrated that cardiomyocyte-specific deletion of Prdm16 during cardiac development results in contractile dysfunction and abnormal electrophysiology of the postnatal heart, resulting in premature death. By combined RNA+ATAC single-cell sequencing, we found that PRDM16 favors ventricular working cardiomyocyte identity, by opposing the activity of master regulators of ventricular conduction and atrial fate. Myocardial loss of PRDM16 during development resulted in hyperplasia of the (distal) ventricular conduction system. Hence, PRDM16 plays an indispensable role during cardiac development by driving ventricular working cardiomyocyte identity.

Unraveling MLL1-fusion leukemia: Epigenetic revelations from an iPS cell point mutation

Our understanding of acute leukemia pathology is heavily dependent on 11q23 chromosomal translocations involving the mixed lineage leukemia-1 (MLL1) gene, a key player in histone H3 lysine 4 (H3K4) methylation. These translocations result in MLL1-fusion (MLL1F) proteins that are thought to drive leukemogenesis. However, the mechanism behind increased H3K4 trimethylation in MLL1F-leukemic stem cells (MLL1F-LSCs), following loss of the catalytic SET domain of MLL1 (known for H3K4 mono- and dimethylation), remains unclear. In our investigation, we introduced a homozygous loss-of-function point mutation in MLL1 within human induced pluripotent stem cells. This mutation mimics the histone methylation, gene expression, and epithelial-mesenchymal transition (EMT) phenotypes of MLL1F-LSCs- without requiring a translocation or functional wild-type MLL1. The mutation caused a genome-wide redistribution of the H3K4 trimethyl mark and upregulated LSC-maintenance genes like HoxA9-A13, Meis1, and the HOTTIP long non-coding RNA (lncRNA). EMT markers such as ZEB1, SNAI2, and HIC-5 were also increased leading to enhanced cellular migration and invasiveness. These observations underscore the essential role of MLL1's enzymatic activity in restraining the cascade of epigenetic changes associated with the gene-activating H3K4 trimethylation mark, which we show may be catalyzed by mislocalized SETd1a H3K4 trimethyltransferase in the absence of MLL1’s enzymatic activity. Challenging existing models, our findings imply that MLL1F-induced leukemias arise from a dominant-negative impact on MLL1's histone methyltransferase activity. We propose targeting SETd1a in precision medicine as a new therapeutic approach for MLL1-associated leukemias.

Sex-specific modulation of renal epigenetic and injury markers in aging kidney.

Sex differences in renal physiology and pathophysiology are now well established in rodent models and in humans. Epigenetic programming is known to be a critical component of renal injury, as studied mainly in male rodent models; however, not much is known about the impact of biological sex and age on the kidney epigenome. We sought to determine the influence of biological sex and age on renal epigenetic and injury markers, using male and female mice at 4 mo (4M; young), 12 mo (12M), and 24 mo (24M; aged) of age. Females had a significant increase in kidney and body weights and serum creatinine levels and a decrease in serum albumin levels from 4M to 24M of age, whereas minor changes were observed in male mice. Kidney injury molecule-1 levels in serum and renal tissue greatly enhanced from 12M to 24M in both males and females. Circulating histone 3 (H3; damage-associated molecular pattern molecules) levels extensively increased with age; however, males had higher levels than females. Overall, females had markedly high histone acetyltransferase (HAT) activity than age-matched males. Aged mice had decreased HAT activity and increased histone deacetylase activity than sex-matched 12M mice. Aged females had substantially decreased renal H3 methylation at lysine 9 and 27 and histone methyltransferase (HMT) activity than aged male mice. Antiaging protein Klotho levels were significantly higher in young males than age-matched females and decreased substantially with age in males, whereas epigenetic repressor of Klotho, trimethylated H3K27, and its HMT enzyme, enhancer of zeste homolog 2, increased consistently with age in both sexes. Moreover, nuclear translocation and activity of proinflammatory transcription factor nuclear factor-κB (p65) were significantly higher in aged mice. Taken together, our data suggest that renal aging lies in a range between normal and diseased kidneys but may differ between female and male mice, highlighting sex-related differences in the aging process.NEW & NOTEWORTHY Although there is evidence of sex-specific differences in kidney diseases, most preclinical studies have used male rodent models. The clinical data on renal injury have typically not been stratified by sex. Our findings provide convincing evidence of sex-specific differences in age-regulated epigenetic alterations and renal injury markers. This study highlights the importance of including both sexes for better realization of underlying sex differences in signaling mechanisms of aging-related renal pathophysiology.

Diverse RNA Structures Induce PRC2 Dimerization and Inhibit Histone Methyltransferase Activity.

Methyltransferase PRC2 (Polycomb Repressive Complex 2) introduces histone H3K27 trimethylation, a repressive chromatin mark, to tune the differential expression of genes. PRC2 is precisely regulated by accessory proteins, histone post-translational modifications and, notably, RNA. Research on PRC2-associated RNA has mostly focused on the tight-binding G-quadruplex (G4) RNAs, which inhibit PRC2 enzymatic activity in vitro and in cells. Our recent cryo-EM structure provided a molecular mechanism for G4 RNA inactivating PRC2 via dimerization, but it remained unclear how diverse RNAs associate with and regulate PRC2. Here, we show that a single-stranded G-rich RNA and an atypical G4 structure called pUG-fold unexpectedly also mediate near-identical PRC2 dimerization resulting in inhibition of PRC2 methyltransferase activity. The conformational flexibility of arginine-rich loops within subunits EZH2 and AEBP2 of PRC2 can accommodate diverse RNA secondary structures, resulting in protein-RNA and protein-protein interfaces similar to those observed previously with G4 RNA. Furthermore, we address a recent report that failed to detect PRC2-associated RNAs in living cells by demonstrating the insensitivity of PRC2-RNA interaction to photochemical crosslinking. Our results support the significance of RNA-mediated PRC2 regulation by showing that this interaction is not limited to a single RNA secondary structure, consistent with the broad PRC2 transcriptome containing many G-tract RNAs incapable of folding into G4 structures.

Abstract PO1-24-05: EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis

Abstract Background: Metaplastic breast carcinomas (mBrCAs) are a highly aggressive subtype of triple negative breast cancer with histological evidence of deregulated differentiation towards non-glandular components. Previous studies have demonstrated that human mBrCA often exhibit activation of differentiation pathways, including canonical Wnt/b-catenin and EZH2-mediated transcriptional repression. Our lab has identified CCN6 as a tumor suppressor in mBrCA. MMTV-Cre;Ccn6fl/fl (CCN6KO) mice develop spindle mBrCAs and CCN6 is reduced/lost in 68% of human mBrCAs. Recently, we have demonstrated that the tumor suppressor function of CCN6 in spindle mBrCA requires activation of Wnt/b-catenin signaling pathway. Here, we tested the hypothesis that CCN6 KO leads to the upregulation of EZH2 histone methyltransferase promoting mBrCA. Furthermore, we investigated the requirement for the activation of the Wnt/b-catenin pathway in this mechanism. Methods: To test the effect of CCN6/b-catenin on EZH2 gene and protein expression we performed IHC, IF, qRT-PCR, ChIP-Seq, RNA-seq, invasion and adhesion assays, EZH2 reporter assay and immunoblots in mBrCA cell lines and MMTV-Cre;Ccn6fl/fl tumors. To investigate the role of CCN6KO-induced b-catenin activation on EZH2 activity and neoplastic functions we employed three independent approaches: i) Expression of a dominant-negative Tcf4 (dnTcf4) rescued with EZH2-WT, dSET and dNLS domain mutants versus vector in MMTV-Cre;Ccn6fl/fl tumor-derived cells; ii) Expression of a constitutively active mutant (S33Y) b-catenin in concert with treatment with recombinant human CCN6 (rhCCN6; 500 ug/ml) versus control; iii) Syngeneic orthotopic mammary tumor transplants of MMTV-Cre;Ccn6fl/fl were used for in vivo rescue experiments with rhCCN6 or BSA. To assess the therapeutic benefit of inhibiting EZH2 methyltransferase activity, MMTV-Cre;Ccn6fl/fl tumor cells (CCN6KO cells) were implanted orthotopically or intracardially in FVB mice, followed by treatment with EPZ-6438 (a selective EZH2 methyltransferase inhibitor) or vehicle. We tested CCN6, b-catenin, and EZH2 expression by IHC in a cohort of 27 human mBrCA tumor samples. Results: CCN6KO-induced b-catenin/TCF activation mediates EZH2 transcriptional upregulation and the deposition of repressive H3K27me3 in spindle mBrCAs. We found that the invasion program triggered by CCN6KO-induced Wnt/b-catenin requires EZH2 catalytic activity as WT-EZH2 (but not dSET-EZH2) rescued the reduced invasion of CCN6KO-dnTcf4 cells. RNA-seq and 3H3K27 ChIP-seq of CCN6KO-dnTcf4 cells transduced with EZH2-WT or dSET-EZH2 identify specific CCN6KO-b-catenin/Tcf targets that require EZH2 transcriptional repressor function. In vivo, administration of CCN6 protein to MMTV-Cre;Ccn6fl/fl tumor transplants reduces tumor growth and nuclear b-catenin, EZH2 and 3H3K27 in the tumors. Pharmacologic inhibition of EZH2 reduces the growth and metastasis of CCN6KO mBrCA tumors and improves survival. We identify a subset of human spindle mBrCA (54%) that display a CCN6Low/nuclear b-cat/EZH2High phenotype. Conclusion: We found a critical role for EZH2 activation in CCN6-deficient mBrCA tumor phenotypes via b-catenin/TCF Wnt canonical signaling. We demonstrate the effectiveness of pharmacological inhibition of EZH2 methyltransferase activity in reducing primary tumor growth and distant metastasis in mouse models of spindle mBrCA. In clinical samples, low CCN6 is significantly associated with activated b-catenin and high EZH2 in spindle mBrCAs compared to other subtypes. These data reveal a novel tumor suppressor mechanism of CCN6 and provide compelling evidence supporting the potential therapeutic value of CCN6 restoration, b-catenin or EZH2 inhibition as promising approaches for the treatment of spindle mBrCAs. Citation Format: Maria Gonzalez, Ahmad Eido, GIUSEPPINA AUGIMERI, Celina Kleer. EZH2 histone methyltransferase activity promotes spindle metaplastic breast carcinoma metastasis and is induced by CCN6 knockout/b-catenin/TCF axis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-05.

Mixed-Lineage Leukaemia Gene Regulates Glucose-Sensitive Gene Expression and Insulin Secretion in Pancreatic Beta Cells.

The escalating prevalence of diabetes mellitus underscores the need for a comprehensive understanding of pancreatic beta cell function. Interest in glucose effectiveness has prompted the exploration of novel regulatory factors. The myeloid/lymphoid or mixed-lineage leukaemia gene (MLL) is widely recognised for its role in leukemogenesis and nuclear regulatory mechanisms through its histone methyltransferase activity in active chromatin. However, its function within pancreatic endocrine tissues remains elusive. Herein, we unveil a novel role of MLL in glucose metabolism and insulin secretion. MLL knockdown in βHC-9 pancreatic beta cells diminished insulin secretion in response to glucose loading, paralleled by the downregulation of the glucose-sensitive genes SLC2a1 and SLC2a2. Similar observations were made in MLL heterozygous knockout mice (MLL+/-), which exhibited impaired glucose tolerance and reduced insulin secretion without morphological anomalies in pancreatic endocrine cells. The reduction in insulin secretion was independent of changes in beta cell mass or insulin granule morphology, suggesting the regulatory role of MLL in glucose-sensitive gene expression. The current results suggest that MLL interacts with circadian-related complexes to modulate the expression of glucose transporter genes, thereby regulating glucose sensing and insulin secretion. Our findings shed light on insulin secretion control, providing potential avenues for therapeutics against diabetes.

Abstract 2640: HBI-2375, a first-in-class IND ready selective MLL1-WDR5 inhibitor, retains desirable preclinical characteristics in solid tumors and in leukemias to be pursued in clinical studies

Abstract MLL1 (mixed lineage leukemia1) interaction with WDR5 (WD repeat domain 5) is critical for its histone methyl transferase (HMT) activity leading to transcriptional regulation of gene expression. MLL1 dysregulation such as its translocation at chromosome 11q23 results in generation of MLL1 fusion genes and leukemia development. Additionally, recent studies indicated a role for MLL1-WDR5 interaction in solid tumors progression via directly affecting cancer cells or by suppression of tumor immunity. Therefore, MLL1-WDR5 interaction is an important target in the discovery of anti-cancer drugs for both leukemia and solid tumors. We recently identified HBI-2375 as a selective inhibitor of MLL1-WDR5 interaction. HBI-2375 showed potent inhibition of WDR5 (IC50: 4.48nM) in biochemical assay and inhibited MV4-11 cellular proliferation (average IC50: 3.17µM) in CTG assays. In addition, HBI-2375 was stable in liver S9 and whole blood, with moderate to high plasma protein binding rates in multiple species, and acceptable hERG (IC50: 17µM). Furthermore, HBI-2375 displayed reasonable oral PK properties (Cmax, T1/2, AUC, and F%) in mouse, rat and dog and showed good safety profile in GLP tox studies. In pharmacology studies HBI-2375 showed significant dose dependent anti-tumor activity in MV4-11 AML model along with reduction in methylation of H3K4 in the tumors harvested from the treated animals demonstrating the targeted disruption of HMT activity in cancer cells by HBI-2375. Furthermore HBI-2375 in combination with PD-1 mAb significantly inhibited tumor growth in MC38, colorectal tumor model, and in 3LL lung carcinoma syngeneic tumor models. Flow cytometry and IHC showed significant increase in the infiltration of CD8+ CTLs in the combination group further providing immune-driven mechanisms for greater efficacy of combination therapy. Gene expression analysis also identified genes that are involved in promoting immune system activity in the combination group. Tissue distribution studies also showed greater accumulation of HBI-2375 in brain tissue indicative of a potential to target glioma and glioblastoma in clinical studies. Therefore, HBI-2375 represents a first-in-class MLL1-WDR5 inhibitor with promising characteristics for future testing in the clinic. Citation Format: Farbod Shojaei, Che Fang, Jill M. Ricono, Mireille Gillings. HBI-2375, a first-in-class IND ready selective MLL1-WDR5 inhibitor, retains desirable preclinical characteristics in solid tumors and in leukemias to be pursued in clinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2640.

Abstract 3223: Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa)

Abstract Advanced PCa, especially castration-resistant PCa, represents an urgent unmet treatment need. Dysregulation of the polycomb repressive complex 2 (PRC2), which mediates trimethylation of histone H3 at lysine 27 (H3K27me3) and leads to gene silencing, is common in PCa and correlates with poor prognosis. EED, a core PRC2 component, is crucial for histone methyltransferase activity. MDM2, a negative regulator of p53, is often amplified or overexpressed in PCa and associated with metastasis and poor clinical outcomes. We evaluated antitumor activity and molecular mechanisms of EED inhibitor APG-5918/EEDi-5273 and MDM2 selective inhibitor alrizomadlin in preclinical PCa models. Human castration-resistant (22Rv1) and androgen-dependent (LNCaP) PCa cell lines were used in antiproliferation assays. Cell viability was determined by CellTiter-Glo luminescent assays. Western blotting was used to explore mechanisms of action. Subcutaneous 22Rv1 and LNCaP cell line-derived xenograft (CDX) models were established to evaluate antitumor effects of APG-5918 and alrizomadlin in vivo. Single-agent APG-5918 or alrizomadlin had limited inhibition on PCa cell growth; combined, they demonstrated synergistic antitumor activity. In the 22Rv1 CDX model, single-agent alrizomadlin (100 mg/kg) or APG-5918 (100 mg/kg) administered for 26 days exerted no/limited antitumor activity, with treatment-to-control (T/C) values of 93.39% and 49.89%, respectively. In contrast, the doublet enhanced antitumor activity (T/C value, 33.22%), with a synergistic index of 1.40. In the LNCaP CDX model, APG-5918 (100 mg/kg) administered for 28 days exhibited minimal antitumor effects (T/C value on last day of dosing, 80.32%). Alrizomadlin (100 or 50 mg/kg) showed limited antitumor activity (T/C value, 47.61%). Combined treatment significantly enhanced antitumor activity (T/C value, 15.69%), with a synergistic index of 2.44. Regarding putative mechanisms, western blot analyses showed that APG-5918 downregulated oncogenic drivers and DNA methylation factors (UHRF1, DNMT1) and on-target-related pharmacodynamic markers (H3K27me3, EED, EZH2). Alrizomadlin markedly downregulated UHRF1 and DNMT1, and upregulated p53 and p21 expression. Combined treatment further enhanced downregulation of DNMT1, UHRF1, cell cycle pathway proteins (pRb, CDK6), antiapoptotic protein MCL-1, and synergistically increased cleavage of PARP-1, a marker of apoptosis. In summary, we show that targeting EED and MDM2 synergistically enhances antitumor activity in PCa cell lines and CDX models. Mechanistically, this combination likely modulated pathways related to DNA methylation, cell cycle, and apoptosis. The findings provide a scientific rationale for future clinical development of APG-5918 and alrizomadlin for PCa patients. Citation Format: Yan Yin, Yun Yang, Eric Liang, Baisong Li, Hengbang Wang, Dajun Yang, Yifan Zhai. Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3223.

Abstract 6586: ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models

Abstract Polycomb repressive complex 2 (PRC2) dysregulation occurs in multiple tumor types and is associated with poor prognosis in patients with prostate cancer. In patients treated with androgen receptor pathway inhibitors (ARPI), emergence of tumor cell plasticity is associated with resistance to therapy. Dysregulation of epigenetic reprogramming factors, including PRC2, creates an environment that is permissive for such lineage plasticity. Inhibition of the PRC2 complex therefore may provide an opportunity to overcome or prevent the emergence of plasticity in prostate cancer. Notably, emerging clinical trial data has demonstrated the potential of ARPI and PRC2 inhibitor combination therapy to improve outcomes in patients with metastatic prostate cancer. PRC2 tri-methylates histone H3 at lysine 27 (H3K27me3), leading to long-term transcriptional silencing, a key mechanism regulating cellular functions such as cell growth and differentiation. Three core subunits comprise PRC2: the catalytic subunit enhancer of zeste homolog 2 (EZH2), suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). EED is essential for the histone methyltransferase activity of PRC2. First-generation PRC2 inhibitors, which target EZH2, exhibit poor drug properties, drug-drug interaction liabilities and short half-life requiring twice daily dosing at high doses in patients. We developed a second generation PRC2 inhibitor, ORIC-944, an allosteric inhibitor of PRC2 that binds the EED subunit. ORIC-944 is a potent, highly selective, orally bioavailable inhibitor with best-in-class properties. ORIC-944 demonstrated single agent tumor growth inhibition in a spectrum of in vivo prostate cancer models, including AR-positive, AR-mutant, ARv7, ARPI-responsive and ARPI-resistant models. In vitro analysis of the combination of ARPIs and ORIC-944 showed synergy in multiple prostate cancer cells, utilizing BLISS and other quantitative methods. In vivo studies revealed that ORIC-944 demonstrated increased efficacy in combination with ARPI, consistent with the in vitro synergy. RNA-seq analysis of transcriptional changes induced by ORIC-944 provided mechanistic insight into the role of PRC2 in prostate cancer lineage plasticity and combination response. These results position ORIC-944 as a best-in-class PRC2 inhibitor for evaluation in combination with ARPI in patients with metastatic prostate cancer. A phase 1b trial of ORIC-944 is ongoing (NCT05413421). Citation Format: Anneleen Daemen, Natalie Yuen, Amber Wang, Aleksandr Pankov, Livia Ulicna, Chelsea Chen, Frank L. Duong, Jason Long, Matthew A. Marx, James G. Christensen, Lori S. Friedman, Melissa R. Junttila. ORIC-944, a potent and selective allosteric PRC2 inhibitor with best-in-class properties, demonstrates combination synergy with AR pathway inhibitors in prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6586.

H3K4me1 facilitates promoter-enhancer interactions and gene activation during embryonic stem cell differentiation

Histone H3 lysine 4 mono-methylation (H3K4me1) marks poised or active enhancers. KMT2C (MLL3) and KMT2D (MLL4) catalyze H3K4me1, but their histone methyltransferase activities are largely dispensable for transcription during early embryogenesis in mammals. To better understand the role of H3K4me1 in enhancer function, we analyze dynamic enhancer-promoter (E-P) interactions and gene expression during neural differentiation of the mouse embryonic stem cells. We found that KMT2C/D catalytic activities were only required for H3K4me1 and E-P contacts at a subset of candidate enhancers, induced upon neural differentiation.By contrast, a majority of enhancers retained H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, H3K4me1 signals at these KMT2C/D-independent sites were reduced after acute depletion ofKMT2B, resulting in aggravated transcriptional defects. Our observations therefore implicate KMT2B in the catalysis of H3K4me1 at enhancers and provide additional support for an active role of H3K4me1 in enhancer-promoter interactions and transcription in mammalian cells.

Selfish conflict underlies RNA-mediated parent-of-origin effects

Genomic imprinting—the non-equivalence of maternal and paternal genomes—is a critical process that has evolved independently in many plant and mammalian species1,2. According to kinship theory, imprinting is the inevitable consequence of conflictive selective forces acting on differentially expressed parental alleles3,4. Yet, how these epigenetic differences evolve in the first place is poorly understood3,5,6. Here we report the identification and molecular dissection of a parent-of-origin effect on gene expression that might help to clarify this fundamental question. Toxin-antidote elements (TAs) are selfish elements that spread in populations by poisoning non-carrier individuals7–9. In reciprocal crosses between two Caenorhabditis tropicalis wild isolates, we found that the slow-1/grow-1 TA is specifically inactive when paternally inherited. This parent-of-origin effect stems from transcriptional repression of the slow-1 toxin by the PIWI-interacting RNA (piRNA) host defence pathway. The repression requires PIWI Argonaute and SET-32 histone methyltransferase activities and is transgenerationally inherited via small RNAs. Remarkably, when slow-1/grow-1 is maternally inherited, slow-1 repression is halted by a translation-independent role of its maternal mRNA. That is, slow-1 transcripts loaded into eggs—but not SLOW-1 protein—are necessary and sufficient to counteract piRNA-mediated repression. Our findings show that parent-of-origin effects can evolve by co-option of the piRNA pathway and hinder the spread of selfish genes that require sex for their propagation.

Novel molecular aberrations involved in testicular germ cell tumor (TGCT) development and cisplatin resistance.

520 Background: Although highly curable, a proportion of testicular germ cell tumors (TGCT) develop resistance to cisplatin-based chemotherapy with subsequent disease relapses and possible patient death. The causes of disease progression and cisplatin (CDDP) resistance have not yet been well elucidated. Methods: We analyzed 56 samples of 15 patients with advanced and relapsing TGCT including primary tumors, metastases persisting after CDDP-containing chemotherapy, and circulating free tumor DNA (cfDNA) from the disease progression, with gDNA from blood leucocytes used as a background control. The quantity and quality of cfDNA was checked by capillary electrophoresis. All samples were sequenced by whole exome sequencing with SureSelect libraries on Illumina platform. Only mutations that were found in malignant samples or cfDNA with variant allele frequency (VAF) ≥ 5% and at least in 3 reads, not present in gDNA, with a minimum total read depth of 5 quality reads, excluding synonymous variants, were further analyzed. Results: Mutations of 6 genes with a significant role in carcinogenesis and/or testis development were identified in more than one patient: RBMX (in 4 pts), TPTE2 and ANKRD30A (in 3 pts), CDC27, PRAMEF8, and PRDM9 (in 2 pts). All the mutations were missense, with VAF 5 - 86%. They were all detectable in patients’ cfDNA, with increasing VAF during the tumor progression; the VAFs in cfDNA collected at advanced disease stages were even higher than those in primary tumors. PRDM9 mutations were found only in cfDNA from disease progression, but not in the primary testicular tumors. Of the identified genes, only CDC27 mutations have been previously described in TGCT. The genes newly associated with TGCT in this study are involved in genomic stability maintenance ( RBMX), spermiogenesis ( TPTE2), cell proliferation and apoptosis ( PRAMEF8), and several of them show testis-specific expression ( TPTE2, PRAMEF8, ANKRD30A, PRDM9). PRDM9 encodes a zinc-finger protein with histone methyltransferase activity, responsible for H3 methylation during meiosis. As epigenetic changes are supposed to play a crucial role in TGCT pathogenesis and resistance, and aberrant H3 methylation has been previously related to abnormal expression of OCT3/4 transcription factor promoting TGCT development, this gene is a strong candidate for TGCT progression and evolving CDDP resistance. Conclusions: Mutations of 5 novel genes have been identified in association with TGCT progression and cisplatin resistance. Their impact is strongly suggested by their functions in normal and malignant regulatory pathways and testis-specific expression patterns but has to be confirmed in experimental studies. The National Institute for Cancer Research project (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Supported by grants MH CZ - DRO (00064190, 00064203).

SETDB2 interacts with BUBR1 to induce accurate chromosome segregation independently of its histone methyltransferase activity.

SETDB2 is a H3K9 histone methyltransferase required for accurate chromosome segregation. Its H3K9 histone methyltransferase activity was reported to be associated with chromosomes during metaphase. Here, we confirm that SETDB2 is required for mitosis and accurate chromosome segregation. However, these functions are independent of its histone methyltransferase activity. Further analysis showed that SETDB2 can interact with BUBR1, and is required for CDC20 binding to BUBR1 and APC/C complex and CYCLIN B1 degradation. The ability of SETDB2 to regulate the binding of CDC20 to BUBR1 or APC/C complex, and stabilization of CYCLIN B1 are also independent of its histone methyltransferase activity. These results suggest that SETDB2 interacts with BUBR1 to promote binding of CDC20 to BUBR1 and APC3, then degrades CYCLIN B1 to ensure accurate chromosome segregation and mitosis, independently of its histone methyltransferase activity.

Macromolecular Complex Including MLL3, Carabin and Calcineurin Regulates Cardiac Remodeling.

Cardiac hypertrophy is an intermediate stage in the development of heart failure. The structural and functional processes occurring in cardiac hypertrophy include extensive gene reprogramming, which is dependent on epigenetic regulation and chromatin remodeling. However, the chromatin remodelers and their regulatory functions involved in the pathogenesis of cardiac hypertrophy are not well characterized. Protein interaction was determined by immunoprecipitation assay in primary cardiomyocytes and mouse cardiac samples subjected or not to transverse aortic constriction for 1 week. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) experiments were performed on the chromatin of adult mouse cardiomyocytes. We report that the calcium-activated protein phosphatase CaN (calcineurin), its endogenous inhibitory protein carabin, the STK24 (STE20-like protein kinase 3), and the histone monomethyltransferase, MLL3 (mixed lineage leukemia 3) form altogether a macromolecular complex at the chromatin of cardiomyocytes. Under basal conditions, carabin prevents CaN activation while the serine/threonine kinase STK24 maintains MLL3 inactive via phosphorylation. After 1 week of transverse aortic constriction, both carabin and STK24 are released from the CaN-MLL3 complex leading to the activation of CaN, dephosphorylation of MLL3, and in turn, histone H3 lysine 4 monomethylation. Selective cardiac MLL3 knockdown mitigates hypertrophy, and chromatin immunoprecipitation and DNA sequencing analysis demonstrates that MLL3 is de novo recruited at the transcriptional start site of genes implicated in cardiomyopathy in stress conditions. We also show that CaN and MLL3 colocalize at chromatin and that CaN activates MLL3 histone methyl transferase activity at distal intergenic regions under hypertrophic conditions. Our study reveals an unsuspected epigenetic mechanism of CaN that directly regulates MLL3 histone methyl transferase activity to promote cardiac remodeling.

Abstract C071: HBI-2375, a selective inhibitor of MLL1-WDR5 interaction, possesses desirable preclinical characteristics in solid tumors in combination with checkpoint inhibitors and also in leukemias in the future clinical investigations

Abstract MLL1 (mixed lineage leukemia1) interaction with WDR5 (WD repeat domain 5) is critical for its histone methyl transferase (HMT) activity leading to transcriptional regulation of gene expression. MLL1 dysregulation such as its translocation at chromosome 11q23 results in generation of MLL1 fusion genes and leukemia development. Additionally, recent studies indicated a role for MLL1-WDR5 interaction in solid tumors progression via directly affecting cancer cells or by suppression of tumor immunity. Therefore, MLL1-WDR5 interaction is an important target in the discovery of anti-cancer drugs for both leukemia and solid tumors. HUYABIO recently identified HBI-2375 as a selective inhibitor of MLL1-WDR5 interaction. HBI-2375 showed potent inhibition of WDR5 (IC50=4.48 nM) and inhibited MV4-11 cellular proliferation (average IC50=3.15 µM) in CTG assays. In addition, HBI-2375 was stable in liver S9 and whole blood, with moderate to high plasma protein binding rates in multiple species, and acceptable hERG IC50 (17 µM). Furthermore, HBI-2375 displayed reasonable oral PK properties (Cmax, T1/2, AUC, and F%) in mouse and dog. In vivo, HBI-2375 has shown significant dose dependent anti-tumor activity in MV4-11 xenograft AML model along with reduction in methylation of H3K4 in the tumors harvested from the treated animals demonstrating the targeted disruption of HMT activity in cancer cells by HBI-2375. Furthermore HBI-2375 in combination with PD-1 mAb significantly inhibited tumor growth in MC38, colorectal tumor model, and in 3LL lung carcinoma syngeneic tumor models. Flow cytometry and IHC showed significant increase in the infiltration of CD8+ CTLs in the combination group further providing immune-driven mechanisms for greater efficacy of combination therapy. Gene expression analysis also identified genes that are involved in promoting immune system activity in the combination group. Therefore, HBI-2375 may represent a first-in-class MLL1-WDR5 inhibitor in the clinic. Citation Format: Farbod Shojaei, Che Fang, Jill M Ricono, Mireille Gillings. HBI-2375, a selective inhibitor of MLL1-WDR5 interaction, possesses desirable preclinical characteristics in solid tumors in combination with checkpoint inhibitors and also in leukemias in the future clinical investigations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C071.

Histone methyltransferase activity affects metabolism in human cells independently of transcriptional regulation.

The N-terminal tails of eukaryotic histones are frequently posttranslationally modified. The role of these modifications in transcriptional regulation is well-documented. However, the extent to which the enzymatic processes of histone posttranslational modification might affect metabolic regulation is less clear. Here, we investigated how histone methylation might affect metabolism using metabolomics, proteomics, and RNA-seq data from cancer cell lines, primary tumour samples and healthy tissue samples. In cancer, the expression of histone methyltransferases (HMTs) was inversely correlated to the activity of NNMT, an enzyme previously characterised as a methyl sink that disposes of excess methyl groups carried by the universal methyl donor S-adenosyl methionine (SAM or AdoMet). In healthy tissues, histone methylation was inversely correlated to the levels of an alternative methyl sink, PEMT. These associations affected the levels of multiple histone marks on chromatin genome-wide but had no detectable impact on transcriptional regulation. We show that HMTs with a variety of different associations to transcription are co-regulated by the Retinoblastoma (Rb) tumour suppressor in human cells. Rb-mutant cancers show increased total HMT activity and down-regulation of NNMT. Together, our results suggest that the total activity of HMTs affects SAM metabolism, independent of transcriptional regulation.

THU486 Identification Of Metabolic And Molecular Mechanisms Driving Estrogen Receptor Positive Breast Cancer Disparities Using A Machine-Learning Pipeline

Abstract Disclosure: A. Santaliz Casiano: None. D. Mehta: None. H. Patel: None. G. Rauscher: None. J. Kim: None. J.M. Frasor: None. K. Hoskins: None. Background: African American (AA) women in the United States have a 40% higher breast cancer mortality rate compared with Non-Hispanic White (NHW) women. The survival disparity is particularly striking among ER+ breast cancer cases. The purpose of this study is to examine whether there are racial differences in metabolic pathways typically activated in patients with ER+ positive breast cancer. Methods: We collected pretreatment plasma from AA and NHW ER+ breast cancer cases and cancer-free controls to conduct an untargeted metabolomics analysis using gas chromatography mass spectrometry (GC-MS) to identify metabolites that may be altered in the different racial groups. Statistical methods combined with multiple feature selection and prediction models were employed to identify race-specific altered metabolic signatures. This was followed by the identification of altered metabolic pathways with a focus in AA patients with breast cancer. The clinical relevance of the identified pathways was further examined in PanCancer Atlas breast cancer data set from TCGA. Results: We identified differential metabolic signatures between NHW and AA patients. In AA patients, we observed changes in metabolites associated with amino acid metabolism, while fatty acid metabolism was significantly enriched in NHW patients. By mapping these metabolites to potential epigenetic regulatory mechanisms, this study identified significant association with regulators of metabolism such as methionine adenosyltransferase 1A (MAT1A), DNA Methyltransferases and Histone methyltransferases for AA individuals, and Fatty acid Synthase (FASN) and Monoacylglycerol lipase (MGL) for NHW individuals. Specific gene NELFE with histone methyltransferase activity was associated with poor survival exclusively for AA individuals. Conclusion: We employed a comprehensive and novel approach that integrates multiple machine learning methods and statistical methods, coupled with human functional pathway analyses. The metabolic profile of plasma samples identified may help elucidate underlying molecular drivers of disproportionately aggressive ER+ tumor biology in AA women, and may ultimately lead to identification of novel therapeutic targets. To our knowledge, this is a novel finding that describes a link between metabolic alterations and epigenetic regulation in AA breast cancer, and underscores the need for detailed investigations into the biological underpinnings of breast cancer health disparities. Presentation: Thursday, June 15, 2023

The SET and ankyrin domains of the secreted Legionella pneumophila histone methyltransferase work together to modify host chromatin.

Legionella pneumophila is an intracellular bacterium responsible of Legionnaires' disease, a severe pneumonia that is often fatal when not treated promptly. The pathogen's ability to efficiently colonize the host resides in its ability to replicate intracellularly. Essential for intracellular replication is translocation of many different protein effectors via a specialized secretion system. One of them, called RomA, binds and directly modifies the host chromatin at a unique site (tri-methylation of lysine 14 of histone H3 [H3K14me]). However, the molecular mechanisms of binding are not known. Here, we resolve this question through structural characterization of RomA together with the H3 peptide. We specifically reveal an active role of the ankyrin repeats located in its C-terminal in the interaction with the histone H3 tail. Indeed, without the ankyrin domains, RomA loses its ability to act as histone methyltransferase. These results discover the molecular mechanisms by which a bacterial histone methyltransferase that is conserved in L. pneumophila strains acts to modify chromatin.