Search

Protein arginine methyltransferases (PRMT) are a widely expressed class of enzymes responsible for catalyzing arginine methylation on numerous protein substrates. Among them, type I PRMTs are responsible for generating asymmetric dimethylarginine. By controlling multiple basic cellular processes, such as DNA damage responses, transcriptional regulation, and mRNA splicing, type I PRMTs contribute to cancer initiation and progression. A type I PRMT inhibitor, GSK3368715, has been developed and has entered clinical trials for solid and hematologic malignancies. Although type I PRMTs have been reported to play roles in modulating immune cell function, the immunologic role of tumor-intrinsic pathways controlled by type I PRMTs remains uncharacterized. Here, our The Cancer Genome Atlas dataset analysis revealed that expression of type I PRMTs associated with poor clinical response and decreased immune infiltration in patients with melanoma. In cancer cell lines, inhibition of type I PRMTs induced an IFN gene signature, amplified responses to IFN and innate immune signaling, and decreased expression of the immunosuppressive cytokine VEGF. In immunocompetent mouse tumor models, including a model of T-cell exclusion that represents a common mechanism of anti–programmed cell death protein 1 (PD-1) resistance in humans, type I PRMT inhibition increased T-cell infiltration, produced durable responses dependent on CD8+ T cells, and enhanced efficacy of anti–PD-1 therapy. These data indicate that type I PRMT inhibition exhibits immunomodulatory properties and synergizes with immune checkpoint blockade (ICB) to induce durable antitumor responses in a T cell–dependent manner, suggesting that type I PRMT inhibition can potentiate an antitumor immunity in refractory settings.

Supplementary Figure from Inhibiting Type I Arginine Methyltransferase Activity Promotes T Cell–Mediated Antitumor Immune Responses

Supplementary Table from Inhibiting Type I Arginine Methyltransferase Activity Promotes T Cell–Mediated Antitumor Immune Responses

Protein arginine methyltransferases (PRMT) are a widely expressed class of enzymes responsible for catalyzing arginine methylation on numerous protein substrates. Among them, type I PRMTs are responsible for generating asymmetric dimethylarginine. By controlling multiple basic cellular processes, such as DNA damage responses, transcriptional regulation, and mRNA splicing, type I PRMTs contribute to cancer initiation and progression. A type I PRMT inhibitor, GSK3368715, has been developed and has entered clinical trials for solid and hematologic malignancies. Although type I PRMTs have been reported to play roles in modulating immune cell function, the immunologic role of tumor-intrinsic pathways controlled by type I PRMTs remains uncharacterized. Here, our The Cancer Genome Atlas dataset analysis revealed that expression of type I PRMTs associated with poor clinical response and decreased immune infiltration in patients with melanoma. In cancer cell lines, inhibition of type I PRMTs induced an IFN gene signature, amplified responses to IFN and innate immune signaling, and decreased expression of the immunosuppressive cytokine VEGF. In immunocompetent mouse tumor models, including a model of T-cell exclusion that represents a common mechanism of anti–programmed cell death protein 1 (PD-1) resistance in humans, type I PRMT inhibition increased T-cell infiltration, produced durable responses dependent on CD8+ T cells, and enhanced efficacy of anti–PD-1 therapy. These data indicate that type I PRMT inhibition exhibits immunomodulatory properties and synergizes with immune checkpoint blockade (ICB) to induce durable antitumor responses in a T cell–dependent manner, suggesting that type I PRMT inhibition can potentiate an antitumor immunity in refractory settings.

Supplementary Table S1: Mutational status of common oncogenes in CRC and STAD cell lines. Supplementary Figure S1: Mutant PIK3CA does not regulate LSD1 expression. Supplementary Figure S2: DNase signal is not generally depleted at TSS's, and LSD1 does not reduce global H3K4me2. Supplementary Figure S3: RCOR1 is generally not upregulated in PIK3CA mutant versus WT in cancers arising from non-gastrointestinal tissues, and generally does not correlate with LSD1 expression. Supplementary Figure S4: LSD1 KO reduces cell viability over time in HT29 but not SW480 cells. Supplementary Figure S5: Inhibiting AKT does not reduce Snail protein level in PIK3CA WT or kinase domain mutant cells, and the LSD1-AKT-GSK3b-Snail axis is context dependent. Supplementary Figure S6: LSD1 is upregulated in PIK3CA mutants classified as CRC subtype CMS4, where C2 domain mutations are also most common. Supplementary Figure S7: EGF treatment causes morphological changes in HT29 WT but not LSD1 KO cells.

Supplementary Figures S1-S6

Frequency of recurrent mutations in the AML cohort

Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells.Implications:Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.

Contains additional details on bioinformatic analyses, including references for packages used.

Disruption of epigenetic regulation is a hallmark of acute myeloid leukemia (AML), but epigenetic therapy is complicated by the complexity of the epigenome. Herein, we developed a long-term primary AML ex vivo platform to determine whether targeting different epigenetic layers with 5-azacytidine and LSD1 inhibitors would yield improved efficacy. This combination was most effective in TET2mut AML, where it extinguished leukemia stem cells and particularly induced genes with both LSD1-bound enhancers and cytosine-methylated promoters. Functional studies indicated that derepression of genes such as GATA2 contributes to drug efficacy. Mechanistically, combination therapy increased enhancer–promoter looping and chromatin-activating marks at the GATA2 locus. CRISPRi of the LSD1-bound enhancer in patient-derived TET2mut AML was associated with dampening of therapeutic GATA2 induction. TET2 knockdown in human hematopoietic stem/progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Our data provide a basis for rational targeting of cooperating aberrant promoter and enhancer epigenetic marks driven by mutant epigenetic modifiers.Significance:Somatic mutations of genes encoding epigenetic modifiers are a hallmark of AML and potentially disrupt many components of the epigenome. Our study targets two different epigenetic layers at promoters and enhancers that cooperate to aberrant gene silencing, downstream of the actions of a mutant epigenetic regulator.This article is highlighted in the In This Issue feature, p. 813

Supplementary Figure 6A-B from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Table 1 from Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells

Aberrant promoter DNA-hypermethylation and repressive chromatin constitutes a frequent mechanism of gene inactivation in cancer. There is great interest in dissecting the mechanisms underlying this abnormal silencing. Studies have shown changes in the nuclear organization of chromatin in tumor cells as well as the association of aberrant methylation with long-range silencing of neighboring genes. Furthermore, certain tumors show a high incidence of promoter methylation termed as the CpG island methylator phenotype. Here, we have analyzed the role of nuclear chromatin architecture for genes in hypermethylated inactive versus nonmethylated active states and its relation with long-range silencing and CpG island methylator phenotype. Using combined immunostaining for active/repressive chromatin marks and fluorescence in situ hybridization in colorectal cancer cell lines, we show that aberrant silencing of these genes occurs without requirement for their being positioned at heterochromatic domains. Importantly, hypermethylation, even when associated with long-range epigenetic silencing of neighboring genes, occurs independent of their euchromatic or heterochromatic location. Together, these results indicate that, in cancer, extensive changes around promoter chromatin of individual genes or gene clusters could potentially occur locally without preference for nuclear position and/or causing repositioning. These findings have important implications for understanding relationships between nuclear organization and gene expression patterns in cancer. Cancer Res; 70(20); 8015–24. ©2010 AACR.

Supplementary Figure 3C-D from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 3H from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 7E-F from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 7G-H from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 5 from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure Legends 1-9, Methods from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 7A-B from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 7C-D from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Table 3 from Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells

Supplementary Table 1 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figure 8 from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 3 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figure 1 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figure 9 from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 2 from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Table 2 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figures 1-6, Table 1B, 2, 4-6 from Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells

Supplementary Figure 4 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figure 6C from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 1 from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 4 from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

Supplementary Figure 5 from Polycomb CBX7 Promotes Initiation of Heritable Repression of Genes Frequently Silenced with Cancer-Specific DNA Hypermethylation

Supplementary Figure 3E-F from Aberrant Silencing of Cancer-Related Genes by CpG Hypermethylation Occurs Independently of Their Spatial Organization in the Nucleus

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

Activation of the epithelial-to-mesenchymal transition (EMT) program is a critical mechanism for initiating cancer progression and migration. Colorectal cancers contain many genetic and epigenetic alterations that can contribute to EMT. Mutations activating the PI3K/AKT signaling pathway are observed in >40% of patients with colorectal cancer contributing to increased invasion and metastasis. Little is known about how oncogenic signaling pathways such as PI3K/AKT synergize with chromatin modifiers to activate the EMT program. Lysine-specific demethylase 1 (LSD1) is a chromatin-modifying enzyme that is overexpressed in colorectal cancer and enhances cell migration. In this study, we determine that LSD1 expression is significantly elevated in patients with colorectal cancer with mutation of the catalytic subunit of PI3K, PIK3CA, compared with patients with colorectal cancer with WT PIK3CA. LSD1 enhances activation of the AKT kinase in colorectal cancer cells through a noncatalytic mechanism, acting as a scaffolding protein for the transcription-repressing CoREST complex. In addition, growth of PIK3CA-mutant colorectal cancer cells is uniquely dependent on LSD1. Knockdown or CRISPR knockout of LSD1 blocks AKT-mediated stabilization of the EMT-promoting transcription factor Snail and effectively blocks AKT-mediated EMT and migration. Overall, we uniquely demonstrate that LSD1 mediates AKT activation in response to growth factors and oxidative stress, and LSD1-regulated AKT activity promotes EMT-like characteristics in a subset of PIK3CA-mutant cells. Implications: Our data support the hypothesis that inhibitors targeting the CoREST complex may be clinically effective in patients with colorectal cancer harboring PIK3CA mutations.

BackgroundAlthough immunotherapy produced dramatic clinical responses in a certain population of cancer patients, tumor cells can employ a variety of immunosuppressive measures to limit the immunotherapeutic efficacy. This highlights a great need to develop novel strategies to expand the clinical benefits of immunotherapy to a broader population of cancer patients. PRMTs have been described as vital regulators of immune responsive pathways in several cell types, but the immunoregulatory role of Type I PRMTs in the tumor microenvironment remains poorly understood.MethodsIn this study, we analyzed the correlation between Type I PRMT expression levels with the clinical outcome or immune signature. Gene expression changes were evaluated in a panel of immunogenic and non-immunogenic cancer cell lines with Type I PRMT inhibitor treatment. The antitumor and immunological effects of Type I PRMT inhibitor were evaluated in combination with checkpoint blockade in a panel of syngeneic tumor models.ResultsUsing TCGA dataset analysis, increased mRNA expression levels of several Type I PRMTs were associated with poor clinical response and decreased immune infiltration in melanoma patients. Particularly, tumors with high expression of PRMT1, the major Type I PRMTs, displayed significantly reduced relapse-free survival (HR=1.891, p=0.038), and were associated with lower cytolytic score (logFC=-0.875, p=1.49e-08) and lower lymphocyte infiltration score (logFC=-0.783, p=0.00077). RNA-seq results showed that interferon signaling was significantly altered after Type I PRMT inhibitor treatment in 10 of 15 cell lines analyzed, with most related genes showing increased expression. In addition, VEGFA was downregulated by 25% or more in 7/8 human and 3/5 mouse cancer cell lines, and a moderate decrease in chromatin accessibility at the Vegfa promoter was observed in ATAC-seq data. Furthermore, Type I PRMT inhibitor combined with anti-PD1 treatment significantly extended the survival of tumor-bearing mice and delayed tumor growth in a panel of immunocompetent mouse models. Mechanistically, Type I PRMT inhibitor significantly increased the apoptotic sensitivity of tumor cells to autologous tumor-reactive T cells in vitro and the infiltration of total T cells (CD3+) in 3 of 4 tested tumor models and cytotoxic T cells (CD8+) in two tested tumor models in vivo.ConclusionsTaken together, these data indicated that Type I PRMT inhibition exhibits immunomodulatory properties and synergizes with immune checkpoint blockade to induce durable antitumor responses in a T cell dependent manner. This study provides a rationale to combine Type I PRMT inhibitor with immune checkpoint blockade to maximize clinical benefits in cancer patients.AcknowledgementsThe authors would like to thank past and present members of the MDACC TIL lab for tumor/TIL processing and banking.

Disruption of epigenetic regulation is a hallmark of acute myeloid leukemia (AML), but epigenetic therapy is complicated by the complexity of the epigenome. Herein, we developed a long-term primary AML ex vivo platform to determine whether targeting different epigenetic layers with 5-azacytidine and LSD1 inhibitors would yield improved efficacy. This combination was most effective in TET2mut AML, where it extinguished leukemia stem cells and particularly induced genes with both LSD1-bound enhancers and cytosine-methylated promoters. Functional studies indicated that derepression of genes such as GATA2 contributes to drug efficacy. Mechanistically, combination therapy increased enhancer–promoter looping and chromatin-activating marks at the GATA2 locus. CRISPRi of the LSD1-bound enhancer in patient-derived TET2mut AML was associated with dampening of therapeutic GATA2 induction. TET2 knockdown in human hematopoietic stem/progenitor cells induced loss of enhancer 5-hydroxymethylation and facilitated LSD1-mediated enhancer inactivation. Our data provide a basis for rational targeting of cooperating aberrant promoter and enhancer epigenetic marks driven by mutant epigenetic modifiers. Significance: Somatic mutations of genes encoding epigenetic modifiers are a hallmark of AML and potentially disrupt many components of the epigenome. Our study targets two different epigenetic layers at promoters and enhancers that cooperate to aberrant gene silencing, downstream of the actions of a mutant epigenetic regulator. This article is highlighted in the In This Issue feature, p. 813

Epigenetic dysregulation is a common feature of most cancers, often occurring directly through alteration of epigenetic machinery. Over the last several years, a new generation of drugs directed at epigenetic modulators have entered clinical development, and results from these trials are now being disclosed. Unlike first-generation epigenetic therapies, these new agents are selective, and many are targeted to proteins which are mutated or translocated in cancer. This review will provide a summary of the epigenetic modulatory agents currently in clinical development and discuss the opportunities and challenges in their development. As these drugs advance in the clinic, drug discovery has continued with a focus on both novel and existing epigenetic targets. We will provide an overview of these efforts and the strategies being employed. Targeting epigenetic modifications is an effective cancer therapeutic approach and can be combined with other therapeutics for maximal effect.

Most, if not all, human cancers exhibit altered epigenetic signatures that promote aberrant gene expression that contributes to cellular transformation. Historically, attempts to pharmacologically intervene in this process have focused on DNA methylation and histone acetylation. More recently, genome-wide studies have identified histone and chromatin regulators as one of the most frequently dysregulated functional classes in a wide range of cancer types. These findings have provided numerous potential therapeutic targets including many that affect histone methylation. These include histone lysine methyltransferases such as enhancer of zeste homolog 2 and DOT1L, protein arginine methyltransferases such as protein arginine methyltransferase 5, and histone lysine demethylases such as lysine-specific demethylase 1. This review presents the rationale for targeting histone methylation in oncology and provides an update on a few key targets that are being investigated in the clinic.

[Image: see text] We report herein the discovery of isoxazole amides as potent and selective SET and MYND Domain-Containing Protein 3 (SMYD3) inhibitors. Elucidation of the structure–activity relationship of the high-throughput screening (HTS) lead compound 1 provided potent and selective SMYD3 inhibitors. The SAR optimization, cocrystal structures of small molecules with SMYD3, and mode of inhibition (MOI) characterization of compounds are described. The synthesis and biological and pharmacokinetic profiles of compounds are also presented.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance., There is increasing evidence that epigenetic mechanisms contribute to therapeutic resistance in cancer. Here the authors study AML patient samples and a mouse model of non-genetic resistance and find that transcriptional plasticity drives stable epigenetic resistance, and identify regulators of enhancer function as important modulators of resistance.