Epigenetic vulnerabilities of leukemia harboring inactivating EZH2 mutations.

Epigenetic regulators, such as the polycomb repressive complex 2 (PRC2), play a critical role in both normal development and carcinogenesis. Mutations and functional dysregulation of PRC2 complex components, such as EZH2, are implicated in various forms of cancer and associated with poor prognosis. This study investigated the epigenetic vulnerabilities of acute myeloid leukemia (AML) and myelodysplastic/myeloproliferative disorders (MDS/MPN) by performing a chemical probe screen in patient cells. Paradoxically, we observed increased sensitivity to EZH2 and embryonic ectoderm development (EED) inhibitors in AML and MDS/MPN patient cells harboring EZH2 mutations. Expression analysis indicated that EZH2 inhibition elicited upregulation of pathways responsible for cell death and growth arrest, specifically in patient cells with mutant EZH2. The identified EZH2 mutations had drastically reduced catalytic activity, resulting in lower cellular H3K27me3 levels, and were associated with decreased EZH2 and PRC2 component EED protein levels. Overall, this study provides an important understanding of the role of EZH2 dysregulation in blood cancers and may indicate disease etiology for these poor prognosis AML and MDS/MPN cases.

PRMT5 regulates ATF4 transcript splicing and oxidative stress response.

Protein methyltransferase 5 (PRMT5) symmetrically dimethylates arginine residues leading to regulation of transcription and splicing programs. Although PRMT5 has emerged as an attractive oncology target, the molecular determinants of PRMT5 dependency in cancer remain incompletely understood. Our transcriptomic analysis identified PRMT5 regulation of the activating transcription factor 4 (ATF4) pathway in acute myelogenous leukemia (AML). PRMT5 inhibition resulted in the expression of unstable, intron-retaining ATF4 mRNA that is detained in the nucleus. Concurrently, the decrease in the spliced cytoplasmic transcript of ATF4 led to lower levels of ATF4 protein and downregulation of ATF4 target genes. Upon loss of functional PRMT5, cells with low ATF4 displayed increased oxidative stress, growth arrest, and cellular senescence. Interestingly, leukemia cells with EVI1 oncogene overexpression demonstrated dependence on PRMT5 function. EVI1 and ATF4 regulated gene signatures were inversely correlated. We show that EVI1-high AML cells have reduced ATF4 levels, elevated baseline reactive oxygen species and increased sensitivity to PRMT5 inhibition. Thus, EVI1-high cells demonstrate dependence on PRMT5 function and regulation of oxidative stress response. Overall, our findings identify the PRMT5-ATF4 axis to be safeguarding the cellular redox balance that is especially important in high oxidative stress states, such as those that occur with EVI1 overexpression.

Author Correction: Caenorhabditis elegans is a useful model for anthelmintic discovery.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Discovery of a chemical probe for PRDM9.

PRDM9 is a PR domain containing protein which trimethylates histone 3 on lysine 4 and 36. Its normal expression is restricted to germ cells and attenuation of its activity results in altered meiotic gene transcription, impairment of double-stranded breaks and pairing between homologous chromosomes. There is growing evidence for a role of aberrant expression of PRDM9 in oncogenesis and genome instability. Here we report the discovery of MRK-740, a potent (IC50: 80 ± 16 nM), selective and cell-active PRDM9 inhibitor (Chemical Probe). MRK-740 binds in the substrate-binding pocket, with unusually extensive interactions with the cofactor S-adenosylmethionine (SAM), conferring SAM-dependent substrate-competitive inhibition. In cells, MRK-740 specifically and directly inhibits H3K4 methylation at endogenous PRDM9 target loci, whereas the closely related inactive control compound, MRK-740-NC, does not. The discovery of MRK-740 as a chemical probe for the PRDM subfamily of methyltransferases highlights the potential for exploiting SAM in targeting SAM-dependent methyltransferases.

Therapeutic Targeting of RNA Splicing Catalysis through Inhibition of Protein Arginine Methylation.

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.

Targeting bivalency de-represses Indian Hedgehog and inhibits self-renewal of colorectal cancer-initiating cells.

In embryonic stem cells, promoters of key lineage-specific differentiation genes are found in a bivalent state, having both activating H3K4me3 and repressive H3K27me3 histone marks, making them poised for transcription upon loss of H3K27me3. Whether cancer-initiating cells (C-ICs) have similar epigenetic mechanisms that prevent lineage commitment is unknown. Here we show that colorectal C-ICs (CC-ICs) are maintained in a stem-like state through a bivalent epigenetic mechanism. Disruption of the bivalent state through inhibition of the H3K27 methyltransferase EZH2, resulted in decreased self-renewal of patient-derived C-ICs. Epigenomic analyses revealed that the promoter of Indian Hedgehog (IHH), a canonical driver of normal colonocyte differentiation, exists in a bivalent chromatin state. Inhibition of EZH2 resulted in de-repression of IHH, decreased self-renewal, and increased sensitivity to chemotherapy in vivo. Our results reveal an epigenetic block to differentiation in CC-ICs and demonstrate the potential for epigenetic differentiation therapy of a solid tumour through EZH2 inhibition.

Characterization of inv(3) cell line OCI-AML-20 with stroma-dependent CD34 expression.

Acute myeloid leukemia (AML) is a complex, heterogeneous disease with variable outcomes following curative intent chemotherapy. AML with inv(3) is a genetic subgroup characterized by a very low response rate to current induction type chemotherapy and thus has among the worst long-term survivorship of the AMLs. Here, we describe OCI-AML-20, a new AML cell line with inv(3) and deletion of chromosome 7; the latter is a common co-occurrence in inv(3) AML. In OCI-AML-20, CD34 expression is maintained and required for repopulation in vitro and in vivo. CD34 expression in OCI-AML-20 shows dependence on the co-culture with stromal cells. Transcriptome analysis indicates that the OCI-AML-20 clusters with other AML patient data sets that have poor prognosis, as well as other AML cell lines, including another inv(3) line, MUTZ-3. OCI-AML-20 is a new cell line resource for studying the biology of inv(3) AML that can be used to identify potential therapies for this poor outcome disease.

The novel nematicide wact-86 interacts with aldicarb to kill nematodes.

Parasitic nematodes negatively impact human and animal health worldwide. The market withdrawal of nematicidal agents due to unfavourable toxicities has limited the available treatment options. In principle, co-administering nematicides at lower doses along with molecules that potentiate their activity could mitigate adverse toxicities without compromising efficacy. Here, we screened for new small molecules that interact with aldicarb, which is a highly effective treatment for plant-parasitic nematodes whose toxicity hampers its utility. From our collection of 638 worm-bioactive compounds, we identified 20 molecules that interact positively with aldicarb to either kill or arrest the growth of the model nematode Caenorhabditis elegans. We investigated the mechanism of interaction between aldicarb and one of these novel nematicides called wact-86. We found that the carboxylesterase enzyme GES-1 hydrolyzes wact-86, and that the interaction is manifested by aldicarb's inhibition of wact-86's metabolism by GES-1. This work demonstrates the utility of C. elegans as a platform to search for new molecules that can positively interact with industrial nematicides, and provides proof-of-concept for prospective discovery efforts.

Caenorhabditis elegans is a useful model for anthelmintic discovery.

Parasitic nematodes infect one quarter of the world's population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery.

Dafadine inhibits DAF-9 to promote dauer formation and longevity of Caenorhabditis elegans.

The DAF-9 cytochrome P450 is a key regulator of dauer formation, developmental timing and longevity in the nematode Caenorhabditis elegans. Here we describe the first identified chemical inhibitor of DAF-9 and the first reported small-molecule tool that robustly induces dauer formation in typical culture conditions. This molecule (called dafadine) also inhibits the mammalian ortholog of DAF-9(CYP27A1), suggesting that dafadine can be used to interrogate developmental control and longevity in other animals.

Compound prioritization methods increase rates of chemical probe discovery in model organisms.

Preselection of compounds that are more likely to induce a phenotype can increase the efficiency and reduce the costs for model organism screening. To identify such molecules, we screened ~81,000 compounds in Saccharomyces cerevisiae and identified ~7500 that inhibit cell growth. Screening these growth-inhibitory molecules across a diverse panel of model organisms resulted in an increased phenotypic hit-rate. These data were used to build a model to predict compounds that inhibit yeast growth. Empirical and in silico application of the model enriched the discovery of bioactive compounds in diverse model organisms. To demonstrate the potential of these molecules as lead chemical probes, we used chemogenomic profiling in yeast and identified specific inhibitors of lanosterol synthase and of stearoyl-CoA 9-desaturase. As community resources, the ~7500 growth-inhibitory molecules have been made commercially available and the computational model and filter used are provided.