Citrullination and the protein code: crosstalk between post-translational modifications in cancer.

Post-translational modifications (PTMs) of proteins are central to epigenetic regulation and cellular signalling, playing an important role in the pathogenesis and progression of numerous diseases. Growing evidence indicates that protein arginine citrullination, catalysed by peptidylarginine deiminases (PADs), is involved in many aspects of molecular and cell biology and is emerging as a potential druggable target in multiple diseases including cancer. However, we are only just beginning to understand the molecular activities of PADs, and their underlying mechanistic details in vivo under both physiological and pathological conditions. Many questions still remain regarding the dynamic cellular functions of citrullination and its interplay with other types of PTMs. This review, therefore, discusses the known functions of PADs with a focus on cancer biology, highlighting the cross-talk between citrullination and other types of PTMs, and how this interplay regulates downstream biological events. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Long non-coding RNA-derived peptides are immunogenic and drive a potent anti-tumour response.

Protein arginine methyltransferase (PRMT) 5 is over-expressed in a variety of cancers and the master transcription regulator E2F1 is an important methylation target. We have explored the role of PRMT5 and E2F1 in regulating the non-coding genome and report here a striking effect on long non-coding (lnc) RNA gene expression. Moreover, many MHC class I protein-associated peptides were derived from small open reading frames in the lncRNA genes. Pharmacological inhibition of PRMT5 or adjusting E2F1 levels qualitatively altered the repertoire of lncRNA-derived peptide antigens displayed by tumour cells. When presented to the immune system as either ex vivo-loaded dendritic cells or expressed from a viral vector, lncRNA-derived peptides drove a potent antigen-specific CD8 T lymphocyte response, which translated into a significant delay in tumour growth. Thus, lncRNA genes encode immunogenic peptides that can be deployed as a cancer vaccine.

The HDAC inhibitor zabadinostat is a systemic regulator of adaptive immunity.

Protein acetylation plays a key role in regulating cellular processes and is subject to aberrant control in diverse pathologies. Although histone deacetylase (HDAC) inhibitors are approved drugs for certain cancers, it is not known whether they can be deployed in other therapeutic contexts. We have explored the clinical HDAC inhibitor, zabadinostat/CXD101, and found that it is a stand-alone regulator of the adaptive immune response. Zabadinostat treatment increased expression of MHC class I and II genes in a variety of cells, including dendritic cells (DCs) and healthy tissue. Remarkably, zabadinostat enhanced the activity of DCs, and CD4 and CD8 T lymphocytes. Using an antigenic peptide presented to the immune system by MHC class I, zabadinostat caused an increase in antigen-specific CD8 T lymphocytes. Further, mice immunised with covid19 spike protein and treated with zabadinostat exhibit enhanced covid19 neutralising antibodies and an increased level of T lymphocytes. The enhanced humoral response reflected increased activity of T follicular helper (Tfh) cells and germinal centre (GC) B cells. Our results argue strongly that zabadinostat has potential to augment diverse therapeutic agents that act through the immune system.

Immune modulation underpins the anti-cancer activity of HDAC inhibitors.

Aberrant protein acetylation is strongly linked to tumorigenesis, and modulating acetylation through targeting histone deacetylase (HDAC) with small-molecule inhibitors has been the focus of clinical trials. However, clinical success on solid tumours, such as colorectal cancer (CRC), has been limited, in part because the cancer-relevant mechanisms through which HDAC inhibitors act remain largely unknown. Here, we have explored, at the genome-wide expression level, the effects of a novel HDAC inhibitor CXD101. In human CRC cell lines, a diverse set of differentially expressed genes were up- and downregulated upon CXD101 treatment. Functional profiling of the expression data highlighted immune-relevant concepts related to antigen processing and natural killer cell-mediated cytotoxicity. Similar profiles were apparent when gene expression was investigated in murine colon26 CRC cells treated with CXD101. Significantly, these changes were also apparent in syngeneic colon26 tumours growing in vivo. The ability of CXD101 to affect immune-relevant gene expression coincided with changes in the tumour microenvironment (TME), especially in the subgroups of CD4 and CD8 tumour-infiltrating T lymphocytes. The altered TME reflected enhanced antitumour activity when CXD101 was combined with immune checkpoint inhibitors (ICIs), such as anti-PD-1 and anti-CTLA4. The ability of CXD101 to reinstate immune-relevant gene expression in the TME and act together with ICIs provides a powerful rationale for exploring the combination therapy in human cancers.

PRMT5 promotes cancer cell migration and invasion through the E2F pathway.

The pRb-E2F pathway is a critical point of regulation in the cell cycle and loss of control of the pathway is a hallmark of cancer. E2F1 is the major target through which pRb exerts its effects and arginine methylation by PRMT5 plays a key role in dictating E2F1 activity. Here we have explored the functional role of the PRMT5-E2F1 axis and highlight its influence on different aspects of cancer cell biology including viability, migration, invasion and adherence. Through a genome-wide expression analysis, we identified a distinct set of genes under the control of PRMT5 and E2F1, including some highly regulated genes, which influence cell migration, invasio and adherence through a PRMT5-dependent mechanism. Most significantly, a coincidence was apparent between the expression of PRMT5 and E2F1 in human tumours, and elevated levels of PRMT5 and E2F1 correlated with poor prognosis disease. Our results suggest a causal relationship between PRMT5 and E2F1 in driving the malignant phenotype and thereby highlight an important pathway for therapeutic intervention.

Arginine methylation expands the regulatory mechanisms and extends the genomic landscape under E2F control.

E2F is a family of master transcription regulators involved in mediating diverse cell fates. Here, we show that residue-specific arginine methylation (meR) by PRMT5 enables E2F1 to regulate many genes at the level of alternative RNA splicing, rather than through its classical transcription-based mechanism. The p100/TSN tudor domain protein reads the meR mark on chromatin-bound E2F1, allowing snRNA components of the splicing machinery to assemble with E2F1. A large set of RNAs including spliced variants associate with E2F1 by virtue of the methyl mark. By focusing on the deSUMOylase SENP7 gene, which we identified as an E2F target gene, we establish that alternative splicing is functionally important for E2F1 activity. Our results reveal an unexpected consequence of arginine methylation, where reader-writer interplay widens the mechanism of control by E2F1, from transcription factor to regulator of alternative RNA splicing, thereby extending the genomic landscape under E2F1 control.

Functional interplay between E2F7 and ribosomal rRNA gene transcription regulates protein synthesis.

A prerequisite for protein synthesis is the transcription of ribosomal rRNA genes by RNA polymerase I (Pol I), which controls ribosome biogenesis. UBF (upstream binding factor) is one of the main Pol I transcription factors located in the nucleolus that activates rRNA gene transcription. E2F7 is an atypical E2F family member that acts as a transcriptional repressor of E2F target genes, and thereby contributes to cell cycle arrest. Here, we describe an unexpected role for E2F7 in regulating rRNA gene transcription. We have found that E2F7 localises to the perinucleolar region, and further that E2F7 is able to exert repressive effects on Pol I transcription. At the mechanistic level, this is achieved in part by E2F7 hindering UBF recruitment to the rRNA gene promoter region, and thereby reducing rRNA gene transcription, which in turn compromises global protein synthesis. Our results expand the target gene repertoire influenced by E2F7 to include Pol I-regulated genes, and more generally suggest a mechanism mediated by effects on Pol I transcription where E2F7 links cell cycle arrest with protein synthesis.

Linking H1 with chromatin and growth control.

The retinoblastoma protein (pRb) is considered to be one of the key regulators of cell proliferation. Here we describe our recent findings that linker histone H1.2 is an interaction partner for pRb and impacts upon the genome-wide chromatin binding properties of pRb. Consequently, H1.2 influences transcriptional repression and cell cycle control.

Tudor-domain protein PHF20L1 reads lysine methylated retinoblastoma tumour suppressor protein.

The retinoblastoma tumour suppressor protein (pRb) classically functions to regulate early cell cycle progression where it acts to enforce a number of checkpoints in response to cellular stress and DNA damage. Methylation at lysine (K) 810, which occurs within a critical CDK phosphorylation site and antagonises a CDK-dependent phosphorylation event at the neighbouring S807 residue, acts to hold pRb in the hypo-phosphorylated growth-suppressing state. This is mediated in part by the recruitment of the reader protein 53BP1 to di-methylated K810, which allows pRb activity to be effectively integrated with the DNA damage response. Here, we report the surprising observation that an additional methylation-dependent interaction occurs at K810, but rather than the di-methyl mark, it is selective for the mono-methyl K810 mark. Binding of the mono-methyl PHF20L1 reader to methylated pRb occurs on E2F target genes, where it acts to mediate an additional level of control by recruiting the MOF acetyltransferase complex to E2F target genes. Significantly, we find that the interplay between PHF20L1 and mono-methyl pRb is important for maintaining the integrity of a pRb-dependent G1-S-phase checkpoint. Our results highlight the distinct roles that methyl-lysine readers have in regulating the biological activity of pRb.

Linker Histone H1.2 Directs Genome-wide Chromatin Association of the Retinoblastoma Tumor Suppressor Protein and Facilitates Its Function.

The retinoblastoma tumor suppressor protein pRb is a master regulator of cellular proliferation, principally through interaction with E2F and regulation of E2F target genes. Here, we describe the H1.2 linker histone as a major pRb interaction partner. We establish that H1.2 and pRb are found in a chromatin-bound complex on diverse E2F target genes. Interrogating the global influence of H1.2 on the genome-wide distribution of pRb indicated that the E2F target genes affected by H1.2 are functionally linked to cell-cycle control, consistent with the ability of H1.2 to hinder cell proliferation and the elevated levels of chromatin-bound H1-pRb complex, which occur in growth-arrested cells. Our results define a network of E2F target genes as susceptible to the regulatory influence of H1.2, where H1.2 augments global association of pRb with chromatin, enhances transcriptional repression by pRb, and facilitates pRb-dependent cell-cycle arrest.

Potent and Selective KDM5 Inhibitor Stops Cellular Demethylation of H3K4me3 at Transcription Start Sites and Proliferation of MM1S Myeloma Cells.

Methylation of lysine residues on histone tail is a dynamic epigenetic modification that plays a key role in chromatin structure and gene regulation. Members of the KDM5 (also known as JARID1) sub-family are 2-oxoglutarate (2-OG) and Fe2+-dependent oxygenases acting as histone 3 lysine 4 trimethyl (H3K4me3) demethylases, regulating proliferation, stem cell self-renewal, and differentiation. Here we present the characterization of KDOAM-25, an inhibitor of KDM5 enzymes. KDOAM-25 shows biochemical half maximal inhibitory concentration values of <100 nM for KDM5A-D in vitro, high selectivity toward other 2-OG oxygenases sub-families, and no off-target activity on a panel of 55 receptors and enzymes. In human cell assay systems, KDOAM-25 has a half maximal effective concentration of ∼50 µM and good selectivity toward other demethylases. KDM5B is overexpressed in multiple myeloma and negatively correlated with the overall survival. Multiple myeloma MM1S cells treated with KDOAM-25 show increased global H3K4 methylation at transcriptional start sites and impaired proliferation.

CBP/p300 Bromodomains Regulate Amyloid-like Protein Aggregation upon Aberrant Lysine Acetylation.

Lysine acetylation is becoming increasingly recognized as a general biological principle in cellular homeostasis, and is subject to abnormal control in different human pathologies. Here, we describe a global effect on amyloid-like protein aggregation in human cells that results from aberrant lysine acetylation. Bromodomain reader proteins are involved in the aggregation process and, using chemical biology and gene silencing, we establish that p300/CBP bromodomains are necessary for aggregation to occur. Moreover, protein aggregation disturbs proteostasis by impairing the ubiquitin proteasome system (UPS) and protein translation, resulting in decreased cell viability. p300/CBP bromodomain inhibitors impede aggregation, which coincides with enhanced UPS function and increased cell viability. Aggregation of a pathologically relevant form of huntingtin protein is similarly affected by p300/CBP inhibition. Our results have implications for understanding the molecular basis of protein aggregation, and highlight the possibility of treating amyloid-like pathologies and related protein folding diseases with bromodomain inhibitor-based strategies.

TLR Adaptor Protein MYD88 Mediates Sensitivity to HDAC Inhibitors via a Cytokine-Dependent Mechanism.

Histone deacetylase (HDAC) inhibitors have proven useful therapeutic agents for certain hematologic cancers. However, HDAC inhibition causes diverse cellular outcomes, and identification of cancer-relevant pathways within these outcomes remains unresolved. In this study, we utilized an unbiased loss-of-function screen and identified the Toll-like receptor (TLR) adaptor protein MYD88 as a key regulator of the antiproliferative effects of HDAC inhibition. High expression of MYD88 exhibited increased sensitivity to HDAC inhibitors; conversely, low expression coincided with reduced sensitivity. MYD88-dependent TLR signaling controlled cytokine levels, which then acted via an extracellular mechanism to maintain cell proliferation and sensitize cells to HDAC inhibition. MYD88 activity was directly regulated through lysine acetylation and was deacetylated by HDAC6. MYD88 was a component of a wider acetylation signature in the ABC subgroup of diffuse large B-cell lymphoma, and one of the most frequent mutations in MYD88, L265P, conferred increased cell sensitivity to HDAC inhibitors. Our study defines acetylation of MYD88, which, by regulating TLR-dependent signaling to cytokine genes, influences the antiproliferative effects of HDAC inhibitors. Our results provide a possible explanation for the sensitivity of malignancies of hematologic origin to HDAC inhibitor-based therapy. Cancer Res; 76(23); 6975-87. ©2016 AACR.

HDAC Inhibitors.

Lysine acetylation in proteins is one of the most abundant posttranslational modifications in eukaryotic cells. The dynamic homeostasis of lysine acetylation and deacetylation is dictated by the action of histone acetyltransferases (HAT) and histone deacetylases (HDAC). Important substrates for HATs and HDACs are histones, where lysine acetylation generally leads to an open and transcriptionally active chromatin conformation. Histone deacetylation forces the compaction of the chromatin with subsequent inhibition of transcription and reduced gene expression. Unbalanced HAT and HDAC activity, and therefore aberrant histone acetylation, has been shown to be involved in tumorigenesis and progression of malignancy in different types of cancer. Therefore, the development of HDAC inhibitors (HDIs) as therapeutic agents against cancer is of great interest. However, treatment with HDIs can also affect the acetylation status of many other non-histone proteins which play a role in different pathways including angiogenesis, cell cycle progression, autophagy and apoptosis. These effects have led HDIs to become anticancer agents, which can initiate apoptosis in tumor cells. Hematological malignancies in particular are responsive to HDIs, and four HDIs have already been approved as anticancer agents. There is a strong interest in finding adequate biomarkers to predict the response to HDI treatment. This chapter provides information on how to assess HDAC activity in vitro and determine the potency of HDIs on different HDACs. It also gives information on how to analyze cellular markers following HDI treatment and to analyze tissue biopsies from HDI-treated patients. Finally, a protocol is provided on how to detect HDI sensitivity determinants in human cells, based on a pRetroSuper shRNA screen upon HDI treatment.

Regulation of actin nucleation and autophagosome formation.

Autophagy is a process of self-eating, whereby cytosolic constituents are enclosed by a double-membrane vesicle before delivery to the lysosome for degradation. This is an important process which allows for recycling of nutrients and cellular components and thus plays a critical role in normal cellular homeostasis as well as cell survival during stresses such as starvation or hypoxia. A large number of proteins regulate various stages of autophagy in a complex and still incompletely understood series of events. In this review, we will discuss recent studies which provide a growing body of evidence that actin dynamics and proteins that influence actin nucleation play an important role in the regulation of autophagosome formation and maturation.

Abrogation of collagen-induced arthritis by a peptidyl arginine deiminase inhibitor is associated with modulation of T cell-mediated immune responses.

Proteins containing citrulline, a post-translational modification of arginine, are generated by peptidyl arginine deiminases (PAD). Citrullinated proteins have pro-inflammatory effects in both innate and adaptive immune responses. Here, we examine the therapeutic effects in collagen-induced arthritis of the second generation PAD inhibitor, BB-Cl-amidine. Treatment after disease onset resulted in the reversal of clinical and histological changes of arthritis, associated with a marked reduction in citrullinated proteins in lymph nodes. There was little overall change in antibodies to collagen or antibodies to citrullinated peptides, but a shift from pro-inflammatory Th1 and Th17-type responses to pro-resolution Th2-type responses was demonstrated by serum cytokines and antibody subtypes. In lymph node cells from the arthritic mice treated with BB-Cl-amidine, there was a decrease in total cell numbers but an increase in the proportion of Th2 cells. BB-Cl-amidine had a pro-apoptotic effect on all Th subsets in vitro with Th17 cells appearing to be the most sensitive. We suggest that these immunoregulatory effects of PAD inhibition in CIA are complex, but primarily mediated by transcriptional regulation. We suggest that targeting PADs is a promising strategy for the treatment of chronic inflammatory disease.

Citrullination-acetylation interplay guides E2F-1 activity during the inflammatory response.

Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression.

Inhibiting WEE1 Selectively Kills Histone H3K36me3-Deficient Cancers by dNTP Starvation.

Histone H3K36 trimethylation (H3K36me3) is frequently lost in multiple cancer types, identifying it as an important therapeutic target. Here we identify a synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to WEE1 inhibition. We show that RRM2, a ribonucleotide reductase subunit, is the target of this synthetic lethal interaction. RRM2 is regulated by two pathways here: first, H3K36me3 facilitates RRM2 expression through transcription initiation factor recruitment; second, WEE1 inhibition degrades RRM2 through untimely CDK activation. Therefore, WEE1 inhibition in H3K36me3-deficient cells results in RRM2 reduction, critical dNTP depletion, S-phase arrest, and apoptosis. Accordingly, this synthetic lethality is suppressed by increasing RRM2 expression or inhibiting RRM2 degradation. Finally, we demonstrate that WEE1 inhibitor AZD1775 regresses H3K36me3-deficient tumor xenografts.

Generation of a Selective Small Molecule Inhibitor of the CBP/p300 Bromodomain for Leukemia Therapy.

The histone acetyltransferases CBP/p300 are involved in recurrent leukemia-associated chromosomal translocations and are key regulators of cell growth. Therefore, efforts to generate inhibitors of CBP/p300 are of clinical value. We developed a specific and potent acetyl-lysine competitive protein-protein interaction inhibitor, I-CBP112, that targets the CBP/p300 bromodomains. Exposure of human and mouse leukemic cell lines to I-CBP112 resulted in substantially impaired colony formation and induced cellular differentiation without significant cytotoxicity. I-CBP112 significantly reduced the leukemia-initiating potential of MLL-AF9(+) acute myeloid leukemia cells in a dose-dependent manner in vitro and in vivo. Interestingly, I-CBP112 increased the cytotoxic activity of BET bromodomain inhibitor JQ1 as well as doxorubicin. Collectively, we report the development and preclinical evaluation of a novel, potent inhibitor targeting CBP/p300 bromodomains that impairs aberrant self-renewal of leukemic cells. The synergistic effects of I-CBP112 and current standard therapy (doxorubicin) as well as emerging treatment strategies (BET inhibition) provide new opportunities for combinatorial treatment of leukemia and potentially other cancers.

Post-translational control of transcription factors: methylation ranks highly.

Methylation of lysine and arginine residues on histones has long been known to determine both chromatin structure and gene expression. In recent years, the methylation of non-histone proteins has emerged as a prevalent modification which impacts on diverse processes such as cell cycle control, DNA repair, senescence, differentiation, apoptosis and tumourigenesis. Many of these non-histone targets represent transcription factors, cell signalling molecules and tumour suppressor proteins. Evidence now suggests that the dysregulation of methyltransferases, demethylases and reader proteins is involved in the development of many diseases, including cancer, and several of these proteins represent potential therapeutic targets for small molecule compounds, fuelling a recent surge in chemical inhibitor design. Such molecules will greatly help us to understand the role of methylation in both health and disease.