Search

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.

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 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.

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.

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.

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.

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.

Cancer drug development has moved from conventional cytotoxic chemotherapeutics to a more mechanism-based targeted approach towards the common goal of tumour growth arrest. The rapid progress in chromatin research has supplied a plethora of potential targets for intervention in cancer. Here, we focus on the histone deacetylase (HDAC) inhibitors, together with their current status of clinical development and potential utility in cancer therapy. HDACs have been widely implicated in growth and transcriptional control, and inhibition of HDAC activity using small molecules causes apoptosis in tumour cells. We discuss the rationale for the development of HDAC inhibitors as novel anti-cancer agents, the potential clinical application and explore ideas on how we may move towards patient stratification with the possibility of increasing efficacy in the clinic.

The ARF protein product of the ink4a/arf locus is induced by a variety of oncogenic signals. ARF facilitates growth arrest through the p53 pathway by hindering the down-regulation of p53 activity mediated by MDM2, through the formation of a protein complex with MDM2. Here we have explored the possibility that human p14(ARF) activity is integrated with growth regulating pathways other than p53, and report our results that p14(ARF) can control the activity of the E2F transcription factor. p14(ARF) regulates E2F activity in different cell-types, including p53(-/-)/mdm(-/-) MEFs, thus excluding that the effects of p14(ARF) are indirectly caused through MDM2 modulation. p14(ARF) down-regulates E2F-dependent transcription, and in cells undergoing E2F-dependent apoptosis prompts cell cycle arrest. p14(ARF) possesses multiple binding domains for E2F-1, one of which resides within the N-terminal region and coincides with the regulation of E2F activity. A mutational analysis of p14(ARF) indicates that the E2F-1 and MDM2 binding domains can be distinguished. These results highlight the potential interplay between p14(ARF) and E2F, and establish p14(ARF) as a pleiotrophic regulator of cell growth that acts by targetting at least two key pathways in the control of proliferation, namely E2F and p53.

Introduction Histone deacetylase inhibitors (HDI) are promising new agents for the treatment of haematological malignancy including lymphoma. HDI have potent anti-proliferative effects in cell-based studies; in the clinical setting, however, the picture is quite variable, and the outcome of HDI therapy is complex and difficult to predict. The toll-like receptor (TLR) adaptor protein MyD88 (Myeloid Differentiation Primary Response Gene 88) is expressed at high levels in haematopoietic tissues and has recently been found to be the target for somatic mutation in lymphoid malignancies. In particular, substitution of the amino acid leucine to proline at position 265 (MyD88 L265P mutation) is estimated to be prevalent in over 90% of cases of Waldenstrom's macroglobulinaemia and around 37% of diffuse large B cell lymphoma (DLBCL) of the ABC subtype. Methods and Results Using an unbiased genome-wide loss-of-function screen (Fotheringham et al, 2009), MyD88 was identified as a key regulator and candidate biomarker which mediates the susceptibility of cancer cells to HDI: We show here in mechanism of action studies that expression of MyD88 can influence sensitivity of various cancer cell lines to treatment with an HDI. Short-term siRNA silencing of MyD88 reduced HDI sensitivity of U2OS osteosarcoma cells (as assessed by immunoblotting and FACS/propidium iodide staining), whereas over-expression of MyD88 and MyD88 L265P led to enhanced sensitivity. Given its role in TLR-signalling, we measured NFκB activity and pro-inflammatory cytokine expression, which were shown to be increased (in particular IL6 levels) when MyD88 was stably or transiently overexpressed in U2OS cells; co-expression of mutated MyD88 L265P augmented this effect. In DLBCL lymphoma cell lines of the ABC subtype, those cells expressing endogenous wild-type MyD88 (RIVA) were found to be less sensitive to HDI treatment compared to those cells carrying a heterozygous MyD88 L265P mutation (HBL-1). MyD88-mutated cells again showed higher levels of IL6 expression on cytokine analysis. Transient knockdown of endogenous MyD88 in ABC DLBCL cells resulted in decreased HDI sensitivity, as did disruption of the TLR/MyD88/NFκB signalling pathway by blocking either the TLR receptor, MyD88 aggregation or IL6 secretion. Immunohistochemical analysis of tumour tissue microarrays from patients with DLBCL carried out in conjunction with this work showed higher expression of MyD88 compared to patients with prostate or colorectal carcinoma. Finally, using a mass spectroscopy approach, we were able to show that MyD88 is acetylated, and that it can be directly deacetylated by the cytoplasmic histone deacetylase HDAC6. Discussion and translational significance Collectively our findings show that in cultured cells, high levels of MyD88 expression are associated with increased sensitivity to HDAC inhibition, whereas low level expression coincides with decreased sensitivity. We hypothesize that MyD88, in its role as adaptor protein in TLR signalling, plays an important role in regulating the expression and autocrine action of pro-inflammatory cytokines such as IL6 which contribute to the sensitization of cancer cells to HDI. The central role played by MyD88 in this mechanism highlights the importance of TLR signalling and innate immunity, and more generally the inflammatory properties of the extra-cellular tumour microenvironment in mediating the effects of HDAC inhibitors on malignant cells. Therefore, both expression level of MyD88 and MyD88 mutation status could serve as a potential biomarker for HDI sensitivity in patient selection. Disclosures No relevant conflicts of interest to declare.

Abnormal epigenetic control is a common early event in tumour progression, and aberrant acetylation in particular has been implicated in tumourigenesis. One of the most promising approaches towards drugs that modulate epigenetic processes has been seen in the development of inhibitors of histone deacetylases (HDACs). HDACs regulate the acetylation of histones in nucleosomes, which mediates changes in chromatin conformation, leading to regulation of gene expression. HDACs also regulate the acetylation status of a variety of other non-histone substrates, including key tumour suppressor proteins and oncogenes. Histone deacetylase inhibitors (HDIs) are potent anti-proliferative agents which modulate acetylation by targeting histone deacetylases. Interest is increasing in HDI-based therapies and so far, two HDIs, vorinostat (SAHA) and romidepsin (FK228), have been approved for treating cutaneous T-cell lymphoma (CTCL). Others are undergoing clinical trials. Treatment with HDIs prompts tumour cells to undergo apoptosis, and cell-based studies have shown a number of other outcomes to result from HDI treatment, including cell-cycle arrest, cell differentiation, anti-angiogenesis and autophagy. However, our understanding of the key pathways through which HDAC inhibitors affect tumour cell growth remains incomplete, which has hampered progress in identifying malignancies other than CTCL which are likely to respond to HDI treatment.

Background: Loss of the histone mark H3 Lysine 36 trimethylation (H3K36me3) is highly prevalent and is associated with poor prognosis. However, there is currently no therapy targeting H3K36me3-deficient cancers. Recently we found a novel synthetic lethal interaction in which H3K36me3-deficient cancers are acutely sensitive to inhibition of WEE1 (a checkpoint kinase). Here we provide our latest and detailed understanding of the underlying mechanism and report our findings in biomarker development. Methods: SETD2 is the sole methyltransferase responsible for generating H3K36me3. We used CRISPR-Cas9 methodology to knockout expression of SETD2 in order to create isogenic cancer cell lines that are negative for H3K36me3. Survival of these isogenic cell lines was measured after treatment with the WEE1 inhibitor AZD1775. Recruitment of transcription factors to the chromatin was analyzed by chromatin immunoprecipitation (ChIP). Rescue experiments were performed by expressing a mutant ribonucleotide reductase (RRM2) that cannot be phosphorylated by CDK (Cyclin-dependent kinase) and is resistant to subsequent degradation by SCF(CyclinF). Immunohistochemistry (IHC) was performed on patient tissue microarrays (>100 samples per cancer type) using a monoclonal antibody against H3K36me3. Results: We showed that SETD2 CRISPR knockout cells are 12-fold more sensitive to the WEE1 inhibitor AZD1775 than parental cells (IC50 = 10 nM vs. 128 nM, p Conclusions: Our study has uncovered the mechanism for the selective killing of H3K36me3-deficient cancers by inhibition of WEE1. We identified novel roles for histone H3K36me3 in promoting gene transcription and DNA replication, and for WEE1 in maintaining nucleotide pools by controlling RRM2 degradation. Our proposed treatment of H3K36me3-deficient cancers is based on synthetic lethality, which provides a less toxic and more effective treatment as it specifically targets cancer cells. Our biomarker analyses revealed that H3K36me3 loss is highly prevalent in renal, colorectal and lung cancers, suggesting that it could be an important therapeutic target in these cancers. H3K36me3 loss can be used as a predictive biomarker for WEE1 inhibitor treatment, and may enable patient selection through immunohistochemistry. Finally, as the WEE1 inhibitor AZD1775, for which we describe a new target, is in clinical trials, we anticipate that these findings will be of immediate clinical relevance. Citation Format: Sophia X. Pfister, Enni Markkanen, Yanyan Jiang, Sovan Sarkar, Mick Woodcock, Lykourgos-Panagiotis Zalmas, Giulia Orlando, Neele Drobnitzky, Grigory Dianov, Songmin Ying, Nicholas B. La Thangue, Vincenzo D'Angiolella, Anderson Ryan, Timothy C. Humphrey. WEE1 inhibition selectively kills histone H3K36me3-deficient cancers by dNTP starvation. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B40.

Histone deacetylase (HDAC) inhibitors are emergent cancer drugs. HR23B is a candidate cancer biomarker identified in a genome-wide loss-of-function screen which influences sensitivity to HDAC inhibitors. Because HDAC inhibitors have found clinical utility in cutaneous T-cell lymphoma (CTCL), we evaluated the role of HR23B in CTCL cells. Our results show that HR23B governs the sensitivity of CTCL cells to HDAC inhibitors. Furthermore, proteasome activity is deregulated in HDAC inhibitor-treated CTCL cells through a mechanism dependent upon HR23B, and HDAC inhibitors sensitize CTCL cells to the effects of proteasome inhibitors. The predictive power of HR23B for clinical response to HDAC inhibitors was investigated through an analysis of a unique collection of CTCL biopsies taken from a phase II clinical trial, where there was a frequent coincidence between HR23B expression and clinical response to HDAC inhibitor. Our study supports the personalized medicine approach for treating cancer and the increasing drive to translate laboratory-based findings into clinical utility.

Post-translational modifications of histone and non-histone proteins by acetylation are known to play a key role in tumourigenesis. Pharmacological manipulation of acetylation has been possible with the identification of small molecule inhibitors of histone deacetylases (HDAC), the enzymes responsible for deacetylating lysine residues. An explosion of drug discovery efforts in recent years has led to the development of an extensive group of HDAC inhibitors, many of which have been shown pre-clinically to have potent anti-tumour activity. Clinical trials using these agents are now underway, with Vorinostat (suberoylanilide hydroxamic acid) having been approved by the FDA for treating cutaneous T-cell lymphoma (CTCL) in patients with progressive, persistent or recurrent disease. This review discusses how biomarkers are being identified and used to expand our knowledge of the mechanisms by which HDAC inhibitors exhibit their anti-cancer effects. In the longer term, biomarkers will provide a means towards achieving patient stratification in tumour types that will respond favourably to HDAC inhibitors.

Although the precise mechanisms by which histone deacetylase inhibitors exert their antitumor activity is not known, certain tumor types undergo a favorable response and consequently several agents have now entered clinical trials. Khan and La Thangue discuss recent developments in our understanding of the molecular events that underlie the anticancer effects of histone deacetylase inhibitors and relate this information to the emerging clinical picture for cutaneous T-cell lymphoma and related malignancies. Reversible acetylation is mediated by histone deacetylase (HDAC), which is involved in regulating a broad repertoire of physiological processes, many of which are under aberrant control in tumor cells. Inhibition of HDAC activity prompts tumor cells to enter apoptosis; therefore, the utility of HDAC inhibitors for the treatment of cancer has been investigated and several HDAC inhibitors have now entered clinical trials. Although the clinical picture is evolving and the precise clinical utility of HDAC inhibitors remains to be determined, it is noteworthy that certain tumor types have a favorable response to such agents. Hematological malignancies seem to be particularly sensitive, and vorinostat (also called suberoylanilide hydroxamic acid) has recently been approved for the treatment of cutaneous manifestations of cutaneous T-cell lymphoma in patients with progressive, persistent or recurrent disease. There are considerable gaps in our understanding of how HDAC inhibitors exert their antitumor activity. In the absence of mechanistic insights into the apoptotic process or biomarkers that inform on responsive tumors, it is a challenge to predict tumor response to HDAC-inhibitor-based therapies with any degree of certainty. In this Review, we discuss recent developments in the understanding of the molecular events that underlie the anticancer effects of HDAC inhibitors, and relate this information to the emerging clinical picture for the treatment of cutaneous T-cell lymphoma and related malignancies.

The p53 tumor suppressor protein is a DNA damage responsive transcription factor that affects diverse cellular processes which include transcription, DNA synthesis and repair, cell cycle arrest, senescence and apoptosis. The Mdm2 oncoprotein is a primary regulator of p53, mediating p53 control via ubiquitin-dependent proteasomal degradation. During DNA damage, the interaction between p53 and Mdm2 is reduced, which allows p53 levels to accumulate. p53 activity is tightly controlled and regulated at a multiplicity of levels, and the importance of co-factors that influence p53 activity is becoming increasingly evident. Recent studies have highlighted the role of Mdm2 in the control of p53 co-factors. Thus, Mdm2 targets JMY, a p53 co-factor, for ubiquitin-dependent Mdm2 targets JMY, a p53 co-factor, for ubiquitin-dependent proteasomal degradation and in doing so overcomes the ability of JMY to augment the p53 response. These results define a new functional relationship between control of p53 activity and Mdm2, and suggest that transcription co-factors which facilitate the p53 response are important targets through which Mdm2 mediates its oncogenic activity.

The E2F1 transcription factor is a recognized regulator of the cell cycle as well as a potent mediator of DNA damage-induced apoptosis and the checkpoint response. Understanding the diverse and seemingly dichotomous functions of E2F1 activity has been the focus of extensive ongoing research. Although the E2F pathway is frequently deregulated in cancer, the contributions of E2F1 itself to tumorigenesis, as a promoter of proliferation or cell death, are far from understood. In this review we aim to provide an update on our current understanding of E2F1, with particular insight into its novel interaction partners and post-translational modifications, as a means to explaining its diverse functional complexity.

E2F is a family of transcription factors implicated in cell cycle control. To understand the role of E2F in controlling cell cycle progression, it is necessary to clarify the breadth of the E2F family. To date, seven E2F subunits have been identified. We report here the characterization of a new E2F subunit, E2F-8, which resembles the organization of E2F-7 in the presence of two separate DNA-binding domains, the integrity of which is required for E2F-8 to bind to DNA. Furthermore, like E2F-7, we find that E2F-8 can repress transcription and delay cell cycle progression. The similarities between E2F-7 and E2F-8 define a new subgroup of the E2F family, and further imply that E2F-7 and E2F-8 may act through overlapping mechanisms in mediating cell cycle control.

The E2F family of transcription factors play an important role in regulating cell cycle progression. We report here the characterization and functional properties of a new member of the human E2F family, referred to as E2F-7. E2F-7 has two separate DNA-binding domains, a feature that distinguishes E2F-7 from other mammalian E2F proteins, but resembling the organization of recently isolated E2F-like proteins from Arabidopsis. E2F-7 binds to DNA independently of a DP partner and delays cell cycle progression. Interestingly, E2F-7 modulates the transcription properties of other E2F proteins. A mutational analysis indicates that the integrity of both DNA-binding domains is required for cell cycle delay and transcriptional modulation. Biochemical results and protein modelling studies suggest that in binding to DNA interactions occur between the two DNA-binding domains, most probably as a homodimer, thereby mimicking the organization of an E2F/DP heterodimer. These structural and functional properties of E2F-7 imply a unique role in regulating cellular proliferation.

The E2F family of transcription factors plays a central role in regulating cellular proliferation by controlling the expression of both the genes required for cell cycle progression, particularly DNA synthesis, and the genes involved with apoptosis. E2F is regulated in a cell cycle-dependent manner, principally through its temporal association with pocket protein family members, the prototype member being the retinoblastoma tumor suppressor protein. Pocket proteins are, in turn, regulated through phosphorylation by cyclin-dependent kinase (cdk). The kinase activity of cyclin/cdk complexes is negatively regulated by cdk inhibitors, and thus both positive and negative growth regulatory signals impinge on E2F activity. Different E2F family members exhibit distinct cell cycle and apoptotic activities. Thus, E2F appears to play a pivotal role in coordinating events connected with proliferation, cell cycle arrest, and apoptosis.

E2F integrates and coordinates cell cycle progression with the transcription apparatus through its cyclical interactions with important regulators of cellular proliferation, such as pRb, cyclins, and cdk's. Physiological E2F is a heterodimeric transcription factor composed of an E2F and a DP family member, and while E2F proteins can stimulate proliferation, certain members of the family are known to be endowed with growth-inhibitory and tumor suppressor-like activity. We have investigated the product of the human mdm2 oncogene, hDM2, and report on its ability to regulate E2F-dependent apoptosis in a fashion that is independent of p53. hDM2 can prevent p53(-/-) cells from entering E2F-dependent apoptosis, an outcome that is dependent upon the presence of the DP subunit. Cells rescued from apoptosis possess lower levels of E2F subunits, although the rescued cells show an increase in DNA synthesis and possess enhanced viability that reflects cooperation between E2F-DP and hMD2. Furthermore, the regulation of E2F activity correlates with an hDM2-dependent effect on the intracellular distribution of DP-1, since hDM2 causes the nuclear accumulation of DP-1. The control of E2F by hDM2 therefore has certain parallels with the targeted degradation by MDM2 of p53. However, the domains in hDM2 required for the regulation of E2F activity can be distinguished from those necessary for p53 degradation, suggesting that control of E2F and p53 by hDM2 may be mechanistically distinct. These experiments define a new level of interplay between E2F and hDM2 whereby hDM2 has a profound impact on the physiological consequences of E2F activation. They suggest that the oncogenic properties of hDM2 may in part be mediated by an antiapoptotic activity that converts E2F from a negative to a positive regulator of cell cycle progression and thereby retains E2F at a level that contributes to a continual state of growth stimulation.

Bcl-2 has previously been characterized as an anti-apoptotic protein. However, it has now been linked to DNA mismatch repair (MMR). By retaining E2F-1 in a transcriptionally inactive state, through the induction of hypophosphorylated retinoblastoma protein, Bcl-2 hinders the expression of a key component of mismatch repair, MSH2. This study could therefore help to explain the mutagenicity that is associated with Bcl-2.

Summary Aberrant acetylation has been strongly linked to tumorigenesis, and the modulation of acetylation through targeting histone deacetylases (HDACs) is gathering increasing pace as a viable therapeutic strategy. A genome-wide loss-of-function screen identified HR23B, which shuttles ubiquitinated cargo proteins to the proteasome, as a sensitivity determinant for HDAC inhibitor-induced apoptosis. HR23B also governs tumor cell sensitivity to drugs that act directly on the proteasome. The level of HR23B influences the response of tumor cells to HDAC inhibitors, and HR23B is found at high levels in cutaneous T cell lymphoma in situ, a malignancy that responds favorably to HDAC inhibitor-based therapy. These results suggest that deregulated proteasome activity contributes to the anticancer activity of HDAC inhibitors.

Besides the genetic information thath is encoded by DNA, heritable information can also be passed on without relying on changes in the nucleotide sequence of DNA, a phenomenon known as epigenetics. Gene expression in eukaryotes is partly regulated by epigenetic mechanisms both at the DNA and histone protein levels. Chromatin structure can be influenced by various modifications, including the reversible posttranslational processes of acetylation and deacetylation of DNA-binding proteins. Histone acetyl transferase (HAT) is referred to as the writer of this process, whereas histone deacetylase (HDAC) is the eraser of this lysine modification. Dysregulation of gene expression and changes in the HDAC expression profile have been associated with carcinogenesis, and HDAC inhibitors are already approved for the treatment of cutaneous T-cell lymphoma and peripheral T-cell lymphoma. These inhibitors are able to influence epigenetic processes by targeting HDAC activity, increasing nuclear histone acetylation status, and contributing to chromatin remodeling, thereby affecting gene expression. In addition, HDACs also act on a plethora of cytosolic proteins with many cellular functions, including angiogenesis, immune responses, and autophagy. In this review, we will give an overview of histone deacetylase and how it can regulate gene expression at the chromatin level.

Summary Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they control important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p107. We now show, using biologically active fusion proteins, that cyclin A can direct the binding of the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p107, as well as activate its histone H1 kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin A-p33cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an activating and targeting subunit of p33cdk2 The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progression and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.