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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.

High-resolution heat map of top gene signatures and GO terms enriched for top MYD88 correlated genes in ABC DLBCL

MYD88 expression and IL-6 sensitises cells to HDAC inhibition

Supplemental figures 1-5 legends

MYD88 expression coincides with an acetylation signature at ABC subgroup-specific GO gene clusters

High levels of MYD88 are apparent in tissue microarray biopsies from patients with DLBCL, but not prostate and colorectal cancer.

HDAC6 is a key regulator of MYD88 acetylation and somatic mutation in MYD88 influences HDI sensitivity.

Supplemental figures 1-5 legends

Description of additional methods and procedures used in the study. Also includes Supplemental References.

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. Cancer Res; 75(23); 5106–19. ©2015 AACR.

BLI evaluation of I-CBP112 selectivity (S1); BioMAP data for I-CBP112 (S2); Effects on MLL-AF9 and NUP98-HOXA9 (S3); Immunophenotype of injected MLL-AF9 cells (S4); Cell survival data (S5); Synergy with JQ1 and doxorubicin (S6).

I-CBP112 ΔTm data for bromodomains (S1); I-CBP112 ITC data (S2); Selectivity screening data outside BRD family (S3); ITC data (S4); Data collection and refinement statistics (S5); I-CBP112 deregulated genes 3d KASUMI-1 (S6); I-CBP112 deregulated genes 3d MOLM-13 (S7); I-CBP112 deregulated genes 3d SEM (S8).

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., CXD101 is a novel histone deacetylase inhibitor with potent antitumour activity. We find that CXD101 reinstates immune‐relevant gene expression in tumours, which includes major histocompatibility complex class I and class II genes. This enables CXD101 to enhance the activity of immune checkpoint therapies, such as anti‐PD‐1, on tumours that would otherwise be poorly responsive and coincides with increased T lymphocyte infiltration into the tumour microenvironment.

Funder: Oxford Experimental Cancer Centre, Background: This Phase 2a dose expansion study was performed to assess the safety, tolerability and preliminary efficacy of the maximum tolerated dose of the oral histone de-acetylase (HDAC) inhibitor CXD101 in patients with relapsed / refractory lymphoma or advanced solid organ cancers and to assess HR23B protein expression by immunohistochemistry as a biomarker of HDAC inhibitor sensitivity. Methods: Patients with advanced solid-organ cancers with high HR23B expression or lymphomas received CXD101 at the recommended phase 2 dose (RP2D). Key exclusions: corrected QT > 450 ms, neutrophils < 1.5 × 109/L, platelets < 75 × 109/L, ECOG > 1. Baseline HR23B expression was assessed by immunohistochemistry. Results: Fifty-one patients enrolled between March 2014 and September 2019, 47 received CXD101 (19 solid-organ cancer, 28 lymphoma). Thirty-four patients received ≥80% RP2D. Baseline characteristics: median age 57.4 years, median prior lines 3, male sex 57%. The most common grade 3–4 adverse events were neutropenia (32%), thrombocytopenia (17%), anaemia (13%), and fatigue (9%) with no deaths on CXD101. No responses were seen in solid-organ cancers, with disease stabilisation in 36% or patients; the overall response rate in lymphoma was 17% with disease stabilisation in 52% of patients. Median progression-free survival was 1.2 months (95% confidence interval (CI) 1.2–5.4) in solid-organ cancers and 2.6 months (95%CI 1.2–5.6) in lymphomas. HR23B status did not predict response. Conclusions: CXD101 showed acceptable tolerability with efficacy seen in Hodgkin lymphoma, T-cell lymphoma and follicular lymphoma. Further studies assessing combination approaches are warranted. Trial registration: ClinicalTrials.gov identifier NCT01977638. Registered 07 November 2013.

Background In the current study, the authors sought to determine the maximum tolerated dose (MTD) of the novel class 1 selective histone deacetylase inhibitor CXD101 in a dose escalation study in patients with advanced solid tumors or recurrent/refractory lymphoma. Methods The authors escalated the dose of CXD101 from 1 mg twice daily orally for 5 days in a 21-day cycle (3+3 design). Results A total of 39 patients were enrolled, 36 of whom received CXD101. Of the 30 patients in the escalation cohort, 29 were evaluable for determination of the dose-limiting toxicity (DLT). DLTs were noted at doses of 16 mg twice daily (1 of 6 patients), 20 mg twice daily (1 of 6 patients), and 24/25 mg twice daily (2 of 5 patients, both of whom developed neutropenic fever). The MTD was 20 mg twice daily, which achieved maximal plasma concentrations (±standard deviation) of 231±76 nM to 342±126 nM, which was within the biologically active range. Six patients received 20 mg twice daily in an expansion cohort. The most frequent adverse events were fatigue, nausea, and reversible cytopenia. Key grade 3 to 4 adverse events (according to Common Terminology Criteria for Adverse Events criteria [version 4.03]) included thrombocytopenia (11%), neutropenia (17%), and neutropenic fever (2%) across the 133 CXD101 cycles given. The toxicity profile was similar to that of licensing studies with other histone deacetylase inhibitors. In 22 evaluable patients receiving a dose of ≥16 mg twice daily (17 of whom had lymphoma and 5 of whom had solid tumors), 3 partial responses (2 in patients with classic Hodgkin lymphoma after allogenic stem cell transplantation and 1 in a patient with angioimmunoblastic T-cell lymphoma) and 1 complete response (in a patient with follicular lymphoma) were noted (overall response rate of 18%) in addition to 9 patients who achieved durable stable disease. Responses were noted predominantly among patients with lymphoma (tumor reduction noted in 63% of patients on standard computed tomography). Conclusions The MTD in the current study was found to be 20 mg twice daily. Encouraging and durable activity was observed in patients with Hodgkin lymphoma, T-cell lymphoma, and follicular lymphoma.

Summary 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., Graphical Abstract, Highlights • Aberrant acetylation causes amyloid-like aggregation and disturbs proteostasis • p300/CBP bromodomain proteins are involved in the protein aggregation • p300/CBP bromodomain inhibitors reverse the cytotoxicity and restore proteostasis • Bromodomain inhibitors also decrease pathological huntingtin protein aggregation, Olzscha et al. demonstrate that aberrantly acetylated proteins form amyloid-like aggregates in human cells. Inhibition of the p300/CBP bromodomains with small-molecule inhibitors reverses the aggregation-induced cytotoxicity. Aggregates caused by a pathologically elongated huntingtin behave in a similar way.

Arginine methylation widens the mechanism of control by E2F1 from a transcription factor to a regulator of alternative RNA splicing., 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.

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.

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. Cancer Res; 75(23); 5106–19. ©2015 AACR.

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.Cell Death and Differentiation advance online publication, 25 August 2017; doi:10.1038/cdd.2017.135.

Over the last 30 years, the transfection of DNA or RNA into mammalian cells has evolved into an ever more sophisticated research tool for the investigation of gene expression and protein function. Transfection is used to describe the process of introducing foreign nucleic acid into a cell. Here we aim to give an overview of commonly used transfection methods, their application, and relative advantages and disadvantages. We will then present a current transfection protocol as a worked example and highlight practical issues such as how to troubleshoot and optimize an existing transfection protocol.

Summary Modulating chromatin through histone methylation orchestrates numerous cellular processes. SETD2-dependent trimethylation of histone H3K36 is associated with active transcription. Here, we define a role for H3K36 trimethylation in homologous recombination (HR) repair in human cells. We find that depleting SETD2 generates a mutation signature resembling RAD51 depletion at I-SceI-induced DNA double-strand break (DSB) sites, with significantly increased deletions arising through microhomology-mediated end-joining. We establish a presynaptic role for SETD2 methyltransferase in HR, where it facilitates the recruitment of C-terminal binding protein interacting protein (CtIP) and promotes DSB resection, allowing Replication Protein A (RPA) and RAD51 binding to DNA damage sites. Furthermore, reducing H3K36me3 levels by overexpressing KDM4A/JMJD2A, an oncogene and H3K36me3/2 demethylase, or an H3.3K36M transgene also reduces HR repair events. We propose that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions promotes cell homeostasis. Moreover, these findings provide insights as to why oncogenic mutations cluster within the H3K36me3 axis., Graphical Abstract, Highlights • A role for SETD2 in DSB resection and homologous recombination repair • Histone H3K36me3 is required for homologous recombination • SETD2 and RAD51 suppress mutations arising from microhomology-mediated end-joining • Mutations affecting H3K36me3 levels may promote tumorigenesis, The SETD2 gene encodes the histone H3K36 trimethyltransferase. Pfister et al. now show that human SETD2-dependent H3K36me3 maintains genome stability by promoting error-free DNA repair through homologous recombination (HR). Upon DNA damage, SETD2-depleted cells exhibit reduced DNA resection, impaired recruitment of early HR factors, and increased utilization of the error-prone microhomology-mediated end-joining repair pathway. Eliminating H3K36me3 by overexpressing the oncogene KDM4A also impairs HR. Thus, H3K36me3 suppresses tumorigenesis by promoting accurate DNA repair.

The mechanisms that underlie and dictate the different biological outcomes of E2F-1 activity have yet to be elucidated. We describe the residue-specific methylation of E2F-1 by the asymmetric dimethylating protein arginine methyltransferase 1 (PRMT1) and symmetric dimethylating PRMT5 and relate the marks to different functional consequences of E2F-1 activity. Methylation by PRMT1 hinders methylation by PRMT5, which augments E2F-1-dependent apoptosis, whereas PRMT5-dependent methylation favors proliferation by antagonizing methylation by PRMT1. The ability of E2F-1 to prompt apoptosis in DNA damaged cells coincides with enhanced PRMT1 methylation. In contrast, cyclin A binding to E2F-1 impedes PRMT1 methylation and augments PRMT5 methylation, thus ensuring that E2F-1 is locked into its cell-cycle progression mode. The Tudor domain protein p100-TSN reads the symmetric methylation mark, and binding of p100-TSN downregulates E2F-1 apoptotic activity. Our results define an exquisite level of precision in the reader-writer interplay that governs the biological outcome of E2F-1 activity. © 2013 Elsevier Inc.

The ubiquitin-like molecule NEDD8 modifies cullin-RING ubiquitin E3 ligases. NEDD8 has been shown to have a few additional substrates, but the extent to which this modification targets non-cullins and the functional significance of such modifications remain unclear. Here, we demonstrate that the cell-cycle-regulating transcription factor E2F-1 is a substrate for NEDD8 post-translational modification. NEDDylation results in decreased E2F-1 stability, lower transcriptional activity and slower cell growth. The lysine residues in E2F-1 targeted for NEDDylation can also be methylated, pointing to a possible interplay between these modifications. These results identify a new mode of E2F-1 regulation and highlight the emerging role of NEDD8 in regulating transcription factor stability and function.

The endocycle represents an alternative cell cycle that is activated in various developmental processes, including placental formation, Drosophila oogenesis, and leaf development. In endocycling cells, mitotic cell cycle exit is followed by successive doublings of the DNA content, resulting in polyploidy. The timing of endocycle onset is crucial for correct development, because polyploidization is linked with cessation of cell division and initiation of terminal differentiation. The anaphase-promoting complex/cyclosome (APC/C) activator genes CDH1 , FZR , and CCS52 are known to promote endocycle onset in human, Drosophila , and Medicago species cells, respectively; however, the genetic pathways governing development-dependent APC/C CDH1/FZR/CCS52 activity remain unknown. We report that the atypical E2F transcription factor E2Fe/DEL1 controls the expression of the CDH1 / FZR orthologous CCS52A2 gene from Arabidopsis thaliana . E2Fe/DEL1 misregulation resulted in untimely CCS52A2 transcription, affecting the timing of endocycle onset. Correspondingly, ectopic CCS52A2 expression drove cells into the endocycle prematurely. Dynamic simulation illustrated that E2Fe/DEL1 accounted for the onset of the endocycle by regulating the temporal expression of CCS52A2 during the cell cycle in a development-dependent manner. Analogously, the atypical mammalian E2F7 protein was associated with the promoter of the APC/C-activating CDH1 gene, indicating that the transcriptional control of APC/C activator genes by atypical E2Fs might be evolutionarily conserved.

Epigenetic proteins are intently pursued targets in ligand discovery. So far, successful efforts have been limited to chromatin modifying enzymes, or so-called epigenetic 'writers' and 'erasers'. Potent inhibitors of histone binding modules have not yet been described. Here we report a cell-permeable small molecule (JQ1) that binds competitively to acetyl-lysine recognition motifs, or bromodomains. High potency and specificity towards a subset of human bromodomains is explained by co-crystal structures with bromodomain and extra-terminal (BET) family member BRD4, revealing excellent shape complementarity with the acetyl-lysine binding cavity. Recurrent translocation of BRD4 is observed in a genetically-defined, incurable subtype of human squamous carcinoma. Competitive binding by JQ1 displaces the BRD4 fusion oncoprotein from chromatin, prompting squamous differentiation and specific antiproliferative effects in BRD4-dependent cell lines and patient-derived xenograft models. These data establish proof-of-concept for targeting protein-protein interactions of epigenetic 'readers', and provide a versatile chemical scaffold for the development of chemical probes more broadly throughout the bromodomain family.

Activation of the p53 tumour suppressor protein in response to DNA damage leads to apoptosis or cell-cycle arrest. Enzymatic modifications are widely believed to affect and regulate p53 activity. We describe here a level of post-translational control that has an important functional consequence on the p53 response. We show that the protein arginine methyltransferase (PRMT) 5, as a co-factor in a DNA damage responsive co-activator complex that interacts with p53, is responsible for methylating p53. Arginine methylation is regulated during the p53 response and affects the target gene specificity of p53. Furthermore, PRMT5 depletion triggers p53-dependent apoptosis. Thus, methylation on arginine residues is an underlying mechanism of control during the p53 response.

The Stress-responsive activator of p300 (Strap) is a transcription co-factor that has an important role in the control of DNA damage response through its ability to regulate p53 activity. Recent studies have however suggested that Strap takes on a wider role in regulating the cellular response to stress. Thus, Strap is now known to be inducible by heat shock and stimulate the transcription of heat-shock genes. A chromatin-associated complex involving heat-shock factor 1 (HSF1), Strap and the p300 co-activator assembles on the HSP70 gene promoter, and Strap augments HSF1 binding and chromatin acetylation in HSP genes, most probably through recruiting p300 acetyltransferase. Cells depleted of Strap do not survive under heat-shock conditions. It appears therefore that Strap is an essential co-factor that acts at the level of chromatin control to regulate heat-shock-responsive transcription. These recent studies underscore the central role taken on by transcription co-factors in integrating and co-ordinating different cellular stress response pathways.

The retinoblastoma protein (pRb) is regarded as the archetypal tumor suppressor and has a vital role in regulating progression through the early stages of the cell cycle.1,2 It does this primarily by binding to and controlling key effectors of cell cycle progression, such as the E2F family of transcription factors.1,2 The pRb-E2F interaction is highly significant since the ability of pRb to bind E2F coincides with growth inhibition and cell cycle delay.2,3 This inhibition is mediated by the reduced transcription of E2F target genes and, reflecting the physiological context in which it occurs, might involve several different mechanisms. For example, pRb downregulates E2F-dependent transcription by its direct association with the transcriptional activation domain of E2F-1.3 pRb can also mediate active repression by recruiting chromatin modifying enzymes such as histone deacetylases and methyltransferases. This enables pRb to transcriptionally silence E2F target genes by influencing the chromatin environment.4 Growth control by pRb is influenced by its post-translational modification, the most widely described to date being phosphorylation by the cyclin-dependent kinases (Cdks). As cells progress through G1, a series of phosphorylation events occur on pRb, and the temporal regulation of pRb phosphorylation reflects the cyclical appearance of cyclin/Cdk complexes.5 Phosphorylation of pRb leads to the inactivation of its tumor suppressor activity, resulting in the release of bound E2F transcription factors and the expression of genes required for cell cycle progression and DNA replication. Phosphorylation, however, is not the only post-translational modification to impinge on pRb activity. For example, pRb is also acetylated under conditions of cell cycle exit, such as differentiation and DNA damage.6,7 Further, pRb can be methylated on a C-terminal lysine residue, and this contributes to transcriptional repression via the recruitment of HP1.8 Methylation of lysine residues is known to control protein activity, and this is particularly clear for transcription factors like p53,9 and in the context of chromatin biology.10 The type of methylation event (mono-, di- or tri-), together with its interplay with other post-translational modifications, can provide a complex level of cross-talk, which can result in the finetuning of a biological response. Indeed, recently, we described a new type of regulation that influences Cdk recognition and phosphorylation of substrate proteins.11 In pRb, a lysine residue located within a region phosphorylated by Cdk kinase was found to be a target for monomethylation by Set7/9, both in vitro and in cells. The lysine residue in question, K810, represents the conserved and essential basic residue found in Cdk substrate proteins (Cdk kinases recognise the concensus sequence S/T-P-X-K/R, where S/T represents the target serine or threonine residue). We demonstrated that methylation at K810 was linked to reduced levels of Cdk phosphorylation at the neighboring serine residues. Interestingly, this interplay between methylation and phosphorylation appeared to be uni-directional, since pRb methylation does not appear to be affected by a prior Cdk phosphorylation event. This suggests that methylation is enforced on the hypophosphorylated, functionally active form of pRb. Indeed, under conditions when pRb is expected to function as a tumor suppressor, such as DNA damage, elevated levels of K810 methylation and a coincident reduction in phosphorylation were apparent. One possible hypothesis for this observed antagonism is that methylation disrupts the interaction between pRb and cyclin/Cdk complexes. This is supported by structural modeling, which suggests that the lysyl side chain of K810 would make a number of important interactions at a cleft formed by the cyclin/Cdk interface, and that these interactions are essential for kinase activity towards the substrate. It is likely that K810 methylation would result in steric clashes with this cyclin/Cdk interface, dislodging the K810 side chain out of its binding pocket. This would reduce the binding efficiency between pRb and cyclin/Cdk and consequently, lead to reduced pRb phosphorylation at the serine sites neighboring K810 (S807/S811). Intriguingly, methylation of K810 also influenced phosphorylation events throughout pRb, even at distant Cdk target sites. This suggests that the ability of Set7/9 to methylate pRb and thereby hinder phosphorylation at S807/S811, subsequently impacts on phosphorylation at other Cdk sites throughout pRb. Whilst the exact mechanism for this global regulation of phosphorylation has yet to be elucidated, a couple of possibilities exist. Firstly, phosphorylation of S807/S811 could represent a priming event that is required for efficient phosphorylation of subsequent Cdk target sites in pRb, perhaps by regulating the recognition by or recruitment of other cyclin/Cdk complexes. There is evidence to suggest, at least, that S807/S811 phosphorylation by cyclin C/Cdk3 occurs earlier in the cell cycle than other cyclin-directed activities, and that this phosphorylation is important for transition of cells from G0 to G1.12 Secondly, methylation of K810 may lead to the recruitment of proteins containing methyl-recognition motifs, which subsequently impact on or interfere with Cdkdependent phosphorylation of pRb. Either way, it is clear that pRb methylation has an important role in regulating cell cycle progression, since disrupting methylation at K810 resulted in decreased G1 arrest, and this was co-incident with decreased association of pRb with the promoters of E2F-1 responsive genes and aberrant transcriptional repression. It is intriguing to see that a type of post-translational code can govern Cdk-dependent growth control. This is another striking example of interplay between two different types of post-translational modification, a feature that is becoming more widely accepted as important for the functional regulation of non-histone proteins. Lysine modification of pRb within a Cdk concensus site provides an alternative and direct substrate-based mechanism to regulate Cdk activity (Fig. 1), which likely acts in concert with other levels of Cdk control such as the induction of Cdk inhibitors like p21.13 Together, both mechanisms may act as a fail-safe to limit pRb phosphorylation by Cdks during stress responses. Since cell cycle arrest in response to stress is primarily a protective mechanism, it seems likely that once this stress is relieved, the methyl mark on pRb would be removed by a demethylation event, permitting cells to re-enter the cell cycle (Fig. 1). It will be of interest not only to identify which demethylase enzyme is involved in this process, but also to revisit other known Cdk substrates and ask whether this methylation-phosphorylation switch is utilized often and in which signaling pathways. Figure 1 Model depicting the effect of methylation on Cdk-dependent growth control. Under conditions of DNA damage, pRb is methylated at K810 by Set7/9. This locks pRb in a hypophosphorylated, growth-arresting state, thereby limiting E2F target gene expression. ...

Summary Actin is an integral component of the cytoskeleton, forming a plethora of macromolecular structures that mediate various cellular functions. The formation of such structures relies on the ability of actin monomers to associate into polymers, and this process is regulated by actin nucleation factors. These factors use monomeric actin pools at specific cellular locations, thereby permitting rapid actin filament formation when required. It has now been established that actin is also present in the nucleus, where it is implicated in chromatin remodelling and the regulation of eukaryotic gene transcription. Notably, the presence of typical actin filaments in the nucleus has not been demonstrated directly. However, studies in recent years have provided evidence for the nuclear localisation of actin nucleation factors that promote cytoplasmic actin polymerisation. Their localisation to the nucleus suggests that these proteins mediate collaboration between the cytoskeleton and the nucleus, which might be dependent on their ability to promote actin polymerisation. The nature of this cooperation remains enigmatic and it will be important to elucidate the physiological relevance of the link between cytoskeletal actin networks and nuclear events. This Commentary explores the current evidence for the nuclear roles of actin nucleation factors. Furthermore, the implication of actin-associated proteins in relaying exogenous signals to the nucleus, particularly in response to cellular stress, will be considered.

We define here a new mechanism through which Mdm2 (mouse double minute 2) regulates p53 activity, by targeting the p53 transcription cofactor JMY. DNA damage causes an increase in JMY protein, and, in a similar manner, small molecule inhibitors of Mdm2 activity induce JMY in unperturbed cells. At a mechanistic level, Mdm2 regulation of JMY requires the Mdm2 RING (really interesting new gene) finger, which promotes the ubiquitin-dependent degradation of JMY. However, regulation of JMY occurs independently of the p53-binding domain in Mdm2 and p53 activity. These results define a new functional relationship between the p53 cofactor JMY and Mdm2, and indicate that transcription cofactors that facilitate p53 activity are important targets for Mdm2 in suppressing the p53 response.

The cellular response to DNA damage, mediated by the DNA repair process, is essential in maintaining the integrity and stability of the genome. E2F-7 is an atypical member of the E2F family with a role in negatively regulating transcription and cell cycle progression under DNA damage. Surprisingly, we found that E2F-7 makes a transcription-independent contribution to the DNA repair process, which involves E2F-7 locating to and binding damaged DNA. Further, E2F-7 recruits CtBP and HDAC to the damaged DNA, altering the local chromatin environment of the DNA lesion. Importantly, the E2F-7 gene is a target for somatic mutation in human cancer and tumor-derived mutant alleles encode proteins with compromised transcription and DNA repair properties. Our results establish that E2F-7 participates in 2 closely linked processes, allowing it to directly couple the expression of genes involved in the DNA damage response with the DNA repair machinery, which has relevance in human malignancy.

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.

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.

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.

The last decade has brought about a vastly improved understanding of the important role that epigenetic changes play in cancer biology. Epigenetic therapy seeks to harness recent insights into the dynamic nature of the mechanisms involved in epigenetic reprogramming in order to develop new therapeutic modalities for the targeted reversal of some of these epigenetic modifications. Already, six epigenetically acting drugs, two DNA methyltransferase inhibitors, and four histone deacetylase inhibitors, have been approved by the US Food and Drug Administration for the treatment of hematological malignancy. However, not all patients benefit from these drugs. Therefore, the development of biomarkers that allow the identification of patients most likely to benefit from these new treatments is a pressing and as yet unmet medical need. Here we discuss recent advances in the development of epigenetic therapies and associated biomarkers and address how these novel modalities open up new perspectives for the future of cancer management in an era of personalized medicine.

The cell cycle constitutes a series of events that lead to the duplication of the cell’s DNA and the generation of two new daughter cells. During the initial stages, the cell grows and expresses numerous genes in preparation for DNA replication, while during the final stages, this newly synthesized DNA is segregated to opposite poles of the cell and the cytoplasm is divided to generate two new daughters. To ensure proper progression from one cell cycle stage to another, the cell employs a number of control mechanisms (known as checkpoints), which constitute a complex set of signalling pathways that respond to both external and internal cues. When these checkpoints function incorrectly, as may occur in response to genetic mutation, the cell cycle control system begins to break down, and this can result in the onset of many human diseases including cancer. Our understanding of the molecular details of cell cycle control is therefore intimately linked with our ability to develop novel cancer therapy and has been a burgeoning area of scientific research for several decades. The molecular details of how the cellular checkpoints function will be summarized within this chapter, with specific examples given of genetic aberrations that compromise this control system, and how such mutations contribute to the onset of tumorigenesis.

Inducing cancer cell death is the basis of the majority of cancer treatments and understanding the mechanisms that control cell death is of prime clinical importance. As a defining feature of cancer is the ability to circumvent cell death pathways, understanding the mechanisms involved is also important in the development of novel therapeutic agents. This chapter outlines three main mechanisms involved in cancer cell death—apoptosis, necroptosis, and autophagy—to give an overview of some of the specific pathways involved. There are a plethora of genetic and epigenetic changes in tumour cells that can circumvent apoptotic pathways; as such understanding and developing therapies that can target other death-signalling pathways could have great clinical significance. Given the complexity involved in the variety of cell death mechanisms, the challenge in oncology is how to harness these different modes of cell death in order to effectively eliminate cancer cells.

Activation of p53 target genes for tumor suppression depends on the stress-specific regulation of transcriptional coactivator complexes. Strap (stress-responsive activator of p300) is activated upon DNA damage by ataxia telangiectasia mutated (ATM) and Chk2 kinases and is a key regulator of the p53 response. In addition to antagonizing Mdm2, Strap facilitates the recruitment of p53 coactivators, including JMY and p300. Strap is a predicted TPR-repeat protein, but shows only limited sequence identity with any protein of known structure. To address this and to elucidate the molecular mechanism of Strap activity we determined the crystal structure of the full-length protein at 2.05 Å resolution. The structure of Strap reveals an atypical six tetratricopeptide repeat (TPR) protein that also contains an unexpected oligonucleotide/oligosaccharide-binding (OB)-fold domain. This previously unseen domain organization provides an extended superhelical scaffold allowing for protein-protein as well as protein-DNA interaction. We show that both of the TPR and OB-fold domains localize to the chromatin of p53 target genes and exhibit intrinsic regulatory activity necessary for the Strap-dependent p53 response.

E2F transcription factors are implicated in diverse cellular functions. The founding member, E2F-1, is endowed with contradictory activities, being able to promote cell-cycle progression and induce apoptosis. However, the mechanisms that underlie the opposing outcomes of E2F-1 activation remain largely unknown. We show here that E2F-1 is directly methylated by PRMT5 (protein arginine methyltransferase 5), and that arginine methylation is responsible for regulating its biochemical and functional properties, which impacts on E2F-1-dependent growth control. Thus, depleting PRMT5 causes increased E2F-1 protein levels, which coincides with decreased growth rate and associated apoptosis. Arginine methylation influences E2F-1 protein stability, and the enhanced transcription of a variety of downstream target genes reflects increased E2F-1 DNA-binding activity. Importantly, E2F-1 is methylated in tumour cells, and a reduced level of methylation is evident under DNA damage conditions that allow E2F-1 stabilization and give rise to apoptosis. Significantly, in a subgroup of colorectal cancer, high levels of PRMT5 frequently coincide with low levels of E2F-1 and reflect a poor clinical outcome. Our results establish that arginine methylation regulates the biological activity of E2F-1 activity, and raise the possibility that arginine methylation contributes to tumourigenesis by influencing the E2F pathway.

Despite its obvious importance in tumorigenesis, little information is available on the mechanisms that integrate cell motility and adhesion with nuclear events. JMY is a transcription co-factor that regulates the p53 response. In addition, JMY contains a series of WH2 domains that facilitate in vitro actin nucleation. We show here that the ability of JMY to influence cell motility is dependent, in part, on its control of cadherin expression as well as the WH2 domains. In DNA damage conditions JMY undergoes nuclear accumulation, which drives the p53 transcription response but reduces its influence on cell motility. Consequently, the role of JMY in actin nucleation is less in damaged cells, although the WH2 domains remain functional in the nucleus where they impact on p53 activity. Together, these findings demonstrate a pathway that links the cytoskeleton with the p53 response, and further suggest that the ability of JMY to regulate actin and cadherin is instrumental in coordinating cell motility with the p53 response.

p53 function is of critical importance in suppressing human cancer formation, highlighted by the fact that the majority of human tumors harbor compromised p53 activity. In normal cells, p53 is held at low levels in a latent form and cellular stress results in the rapid stabilization of p53. Mdm2 mediates ubiquitin-dependent degradation of p53 which plays a key role in maintaining cellular p53 levels. Ubiquitination was, until recently, considered a straightforward system involved in p53 degradation, but recent work has demonstrated how ubiquitination can alter p53 activity, not stability. In this review we summarize current understanding on p53 ubiquitination by Mdm2 with a particular focus on how the balance between protein levels and other post-translational modifications will direct the p53 response.