The child cancer, neuroblastoma (NB), is characterised by a low incidence of mutations and strong oncogenic embryonal driver signals. Many new targeted epigenetic modifier drugs have failed in human trials as monotherapy.
We performed a high-throughput, combination chromatin-modifier drug screen against NB cells. We screened 13 drug candidates in 78 unique combinations.
We found that the combination of two histone methyltransferase (HMT) inhibitors: GSK343, targeting EZH2, and SGC0946, targeting DOT1L, demonstrated the strongest synergy across 8 NB cell lines, with low normal fibroblast toxicity. High mRNA expression of both EZH2 and DOT1L in NB tumour samples correlated with the poorest patient survival. Combination HMT inhibitor treatment caused activation of ATF4-mediated endoplasmic reticulum (ER) stress responses. In addition, glutathione and several amino acids were depleted by HMT inhibitor combination on mass spectrometry analysis. The combination of SGC0946 and GSK343 reduced tumour growth in comparison to single agents.
Our results support further investigation of HMT inhibitor combinations as a therapeutic approach in NB.
© 2024 The Author(s). Cancer Medicine published by John Wiley & Sons Ltd.

All-trans-retinoic acid (ATRA)-based differentiation therapy of acute promyelocytic leukemia (APL) represents one of the most clinically effective examples of precision medicine and the first example of targeted oncoprotein degradation. The success of ATRA in APL, however, remains to be translated to non-APL acute myeloid leukemia (AML). We previously showed that aberrant histone modifications, including histone H3 lysine 4 (H3K4) and lysine 27 (H3K27) methylation, were associated with this lack of response and that epigenetic therapy with small molecule inhibitors of the H3K4 demethylase LSD1/KDM1A could reprogram AML cells to respond to ATRA. Serving as the enzymatic component of Polycomb Repressive Complex 2, EZH2/KMT6A methyltransferase plays a critical role in normal hematopoiesis by affecting the balance between self-renewal and differentiation. The canonical function of EZH2 is methylation of H3K27, although important non-canonical roles have recently been described. EZH2 mutation or deregulated expression has been conclusively demonstrated in the pathogenesis of AML and response to treatment, thus making it an attractive therapeutic target. In this study, we therefore investigated whether inhibition of EZH2 might also improve the response of non-APL AML cells to ATRA-based therapy. We focused on GSK-343, a pyridone-containing S-adenosyl-L-methionine cofactor-competitive EZH2 inhibitor that is representative of its class, and HKMTI-1-005, a substrate-competitive dual inhibitor targeting EZH2 and the closely related G9A/GLP H3K9 methyltransferases. We found that treatment with HKMTI-1-005 phenocopied EZH2 knockdown and was more effective in inducing differentiation than GSK-343, despite the efficacy of GSK-343 in terms of abolishing H3K27 trimethylation. Furthermore, transcriptomic analysis revealed that in contrast to treatment with GSK-343, HKMTI-1-005 upregulated the expression of differentiation pathway genes with and without ATRA, while downregulating genes associated with a hematopoietic stem cell phenotype. These results pointed to a non-canonical role for EZH2, which was supported by the finding that EZH2 associates with the master regulator of myeloid differentiation, RARα, in an ATRA-dependent manner that was enhanced by HKMTI-1-005, possibly playing a role in co-regulator complex exchange during transcriptional activation. In summary, our results strongly suggest that addition of HKMTI-1-005 to ATRA is a new therapeutic approach against AML that warrants further investigation.
Copyright © 2023 Sbirkov, Schenk, Kwok, Stengel, Brown, Brown, Chesler, Zelent, Fuchter and Petrie.

Histone H3 trimethylation at lysine 27 (H3K27me3) is known to act as a transcriptionally repressive histone modification via heterochromatin formation. In skeletal muscle, it was also reported that H3K27me3 was enriched at the sites transcriptionally activated by exercise, although the role of H3K27me3 in adaptation to exercise is unknown. In this study, using mouse tibialis anterior muscle, we initially determined the genome-wide enrichment of RNA polymerase II and histone H3 trimethylation at lysine 4 (H3K4me3) and H3K27me3 using chromatin immunoprecipitation, followed by sequencing analysis. The loci that were transcriptionally upregulated by a single bout of running exercise were marked by both H3K27me3 and H3K4me3, which were also correlated with the distribution of RNA polymerase II. The genes that were not responsive to exercise exhibited high H3K4me3 occupancy, similar to the upregulated genes but with less H3K27me3. Next, we tested the effects of GSK343, a specific inhibitor of enhancer of zeste homologue 2 (EZH2). Unexpectedly, GSK343 administration enhanced the H3K27me3 occupancy at the target loci, leading to the upregulation of gene responses to acute exercise. Administration of GSK343 also facilitated the phenotypic transformation of type IIb to type IIa fibres and the upregulation of AMPK phosphorylation and levels of heat shock protein 70, pyruvate dehydrogenase kinase 4, peroxisome proliferator-activated receptor γ coactivator-1α and muscle RING finger 1. Furthermore, in contrast to the accelerated adaptation to exercise by GSK343, administration of the EZH1/2 dual inhibitor valemetostat prevented the changes in the aforementioned parameters after exercise training. These results indicate that exercise-induced H3K27me3 plays a key role in inducing exercise-related effects in the skeletal muscle. KEY POINTS: Exercise mediates histone H3 trimethylation at lysine 27 (H3K27me3) at transcriptionally upregulated loci in skeletal muscle, but the role of H3K27me3 in the adaptation of skeletal muscle to exercise training is unclear. Chromatin immunoprecipitation followed by sequencing analysis demonstrated that H3K27me3, in addition to H3K4me3 modifications, is the hallmark of sites showing higher responses to acute exercise. GSK343, a selective inhibitor of the enhancer of zeste homologue 2 (EZH2), enhanced the gene responses to a single bout of exercise and accelerated the adaptive changes during exercise training in association with myonuclear H3K27me3 accumulation. Administration of valemetostat, an EZH1/2 dual inhibitor, repressed myonuclear H3K27me3 accumulation during training and caused a failure of adaptive changes. Exercise-induced H3K27me3 might play a key role in inducing exercise-related effects in skeletal muscle.
© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.

Despite significant progress in the treatment of patients with diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), the prognosis of patients with relapsed disease remains poor due to the emergence of drug resistance and subsequent disease progression. Identification of novel targets and therapeutic strategies for these diseases represents an urgent need. Here, we report that both MCL and DLBCL are exquisitely sensitive to transcription-targeting drugs, in particular THZ531, a covalent inhibitor of cyclin-dependent kinase 12 (CDK12). By implementing pharmacogenomics and a cell-based drug screen, we found that THZ531 leads to inhibition of oncogenic transcriptional programs, especially the DNA damage response pathway, MYC target genes and the mTOR-4EBP1-MCL-1 axis, contributing to dramatic lymphoma suppression in vitro. We also identified de novo and established acquired THZ531-resistant lymphoma cells conferred by over-activation of the MEK-ERK and PI3K-AKT-mTOR pathways and upregulation of multidrug resistance-1 (MDR1) protein. Of note, EZH2 inhibitors reversed resistance to THZ531 by competitive inhibition of MDR1 and, in combination with THZ531, synergistically inhibited MCL and DLBCL growth in vitro. Our study indicates that CDK12 inhibitors, alone or together with EZH2 inhibitors, offer promise as novel effective approaches for difficult-to-treat DLBCL and MCL.

Despite viral suppression due to combination antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) continue to affect half of people with HIV, suggesting that certain antiretrovirals (ARVs) may contribute to HAND.
We examined the effects of nucleoside/nucleotide reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) and the integrase inhibitors dolutegravir (DTG) and elvitegravir (EVG) on viability, structure, and function of glutamatergic neurons (a subtype of CNS neuron involved in cognition) derived from human induced pluripotent stem cells (hiPSC-neurons), and primary human neural precursor cells (hNPCs), which are responsible for neurogenesis.
Using automated digital microscopy and image analysis (high content analysis, HCA), we found that DTG, EVG, and TDF decreased hiPSC-neuron viability, neurites, and synapses after 7 days of treatment. Analysis of hiPSC-neuron calcium activity using Kinetic Image Cytometry (KIC) demonstrated that DTG and EVG also decreased the frequency and magnitude of intracellular calcium transients. Longer ARV exposures and simultaneous exposure to multiple ARVs increased the magnitude of these neurotoxic effects. Using the Microscopic Imaging of Epigenetic Landscapes (MIEL) assay, we found that TDF decreased hNPC viability and changed the distribution of histone modifications that regulate chromatin packing, suggesting that TDF may reduce neuroprogenitor pools important for CNS development and maintenance of cognition in adults.
This study establishes human preclinical assays that can screen potential ARVs for CNS toxicity to develop safer cART regimens and HAND therapeutics.
Copyright © 2022 Elsevier Inc. All rights reserved.