Conofolidine: A Natural Plant Alkaloid That Causes Apoptosis and Senescence in Cancer Cells.

Natural products contribute substantially to anticancer therapy; the plant kingdom provides an important source of molecules. Conofolidine is a novel Aspidosperma-Aspidosperma bisindole alkaloid isolated from the Malayan plant Tabernaemontana corymbosa. Herein, we report conofolidine's broad-spectrum anticancer activity together with that of three other bisindoles-conophylline, leucophyllidine, and bipleiophylline-against human-derived breast, colorectal, pancreatic, and lung carcinoma cell lines. Remarkably, conofolidine was able to induce apoptosis (e.g., in MDA-MB-468 breast) or senescence (e.g., in HT-29 colorectal) in cancer cells. Annexin V-FITC/PI, caspase activation, and PARP cleavage confirmed the former while positive ß-gal staining corroborated the latter. Cell cycle perturbations were evident, comprising S-phase depletion, accompanied by downregulated CDK2, and cyclins (A2, D1) with p21 upregulation. Confocal imaging of HCT-116 cells revealed an induction of aberrant mitotic phenotypes-membrane blebbing, DNA-fragmentation with occasional multi-nucleation. DNA integrity assessment in HCT-116, MDA-MB-468, MIAPaCa-2, and HT-29 cells showed increased fluorescent γ-H2AX during the G1 cell cycle phase; γ-H2AX foci were validated in HCT-116 and MDA-MB-468 cells by confocal microscopy. Conofolidine increased oxidative stress, preceding apoptosis- and senescence-induction in most carcinoma cell lines as seen by enhanced ROS levels accompanied by increased NQO1 expression. Collectively, we present conofolidine as a putative potent anticancer agent capable of inducing heterogeneous modes of cancerous cell death in vitro, encouraging further preclinical evaluations of this natural product.

Novel Semi-Synthetic Cu (II)-Cardamonin Complex Exerts Potent Anticancer Activity against Triple-Negative Breast and Pancreatic Cancer Cells via Inhibition of the Akt Signaling Pathway.

Cardamonin is a polyphenolic natural product that has been shown to possess cytotoxic activity against a variety of cancer cell lines. We previously reported the semi-synthesis of a novel Cu (II)-cardamonin complex (19) that demonstrated potent antitumour activity. In this study, we further investigated the bioactivity of 19 against MDA-MB-468 and PANC-1 cancer cells in an attempt to discover an effective treatment for triple-negative breast cancer (TNBC) and pancreatic cancer, respectively. Results revealed that 19 abolished the formation of MDA-MB-468 and PANC-1 colonies, exerted growth-inhibitory activity, and inhibited cancer cell migration. Further mechanistic studies showed that 19 induced DNA damage resulting in gap 2 (G2)/mitosis (M) phase arrest and microtubule network disruption. Moreover, 19 generated reactive oxygen species (ROS) that may contribute to induction of apoptosis, corroborated by activation of caspase-3/7, PARP cleavage, and downregulation of Mcl-1. Complex 19 also decreased the expression levels of p-Akt and p-4EBP1, which indicates that the compound exerts its activity, at least in part, via inhibition of Akt signalling. Furthermore, 19 decreased the expression of c-Myc in PANC-1 cells only, which suggests that it may exert its bioactivity via multiple mechanisms of action. These results demonstrate the potential of 19 as a therapeutic agent for TNBC and pancreatic cancer.

The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification.

Histone tail modifications control many nuclear processes by dictating the dynamic exchange of regulatory proteins on chromatin. Here we report novel insights into histone H3 tail structure in complex with the double PHD finger (DPF) of the lysine acetyltransferase MOZ/MYST3/KAT6A. In addition to sampling H3 and H4 modification status, we show that the DPF cooperates with the MYST domain to promote H3K9 and H3K14 acetylation, although not if H3K4 is trimethylated. Four crystal structures of an extended DPF alone and in complex with unmodified or acetylated forms of the H3 tail reveal the molecular basis of crosstalk between H3K4me3 and H3K14ac. We show for the first time that MOZ DPF induces α-helical conformation of H3K4-T11, revealing a unique mode of H3 recognition. The helical structure facilitates sampling of H3K4 methylation status, and proffers H3K9 and other residues for modification. Additionally, we show that a conserved double glycine hinge flanking the H3 tail helix is required for a conformational change enabling docking of H3K14ac with the DPF. In summary, our data provide the first observations of extensive helical structure in a histone tail, revealing the inherent ability of the H3 tail to adopt alternate conformations in complex with chromatin regulators.

A signature motif mediating selective interactions of BCL11A with the NR2E/F subfamily of orphan nuclear receptors.

Despite their physiological importance, selective interactions between nuclear receptors (NRs) and their cofactors are poorly understood. Here, we describe a novel signature motif (F/YSXXLXXL/Y) in the developmental regulator BCL11A that facilitates its selective interaction with members of the NR2E/F subfamily. Two copies of this motif (named here as RID1 and RID2) permit BCL11A to bind COUP-TFs (NR2F1;NR2F2;NR2F6) and Tailless/TLX (NR2E1), whereas RID1, but not RID2, binds PNR (NR2E3). We confirmed the existence of endogenous BCL11A/TLX complexes in mouse cortex tissue. No interactions of RID1 and RID2 with 20 other ligand-binding domains from different NR subtypes were observed. We show that RID1 and RID2 are required for BCL11A-mediated repression of endogenous γ-globin gene and the regulatory non-coding transcript Bgl3, and we identify COUP-TFII binding sites within the Bgl3 locus. In addition to their importance for BCL11A function, we show that F/YSXXLXXL/Y motifs are conserved in other NR cofactors. A single FSXXLXXL motif in the NR-binding SET domain protein NSD1 facilitates its interactions with the NR2E/F subfamily. However, the NSD1 motif incorporates features of both LXXLL and FSXXLXXL motifs, giving it a distinct NR-binding pattern in contrast to other cofactors. In summary, our results provide new insights into the selectivity of NR/cofactor complex formation.

Correction to "Apoferritin-Encapsulated Jerantinine A for Transferrin Receptor Targeting and Enhanced Selectivity in Breast Cancer Therapy".

[This corrects the article DOI: 10.1021/acsomega.2c00997.].

Differential effects of garcinol and curcumin on histone and p53 modifications in tumour cells.

BACKGROUND: Post-translational modifications (PTMs) of histones and other proteins are perturbed in tumours. For example, reduced levels of acetylated H4K16 and trimethylated H4K20 are associated with high tumour grade and poor survival in breast cancer. Drug-like molecules that can reprogram selected histone PTMs in tumour cells are therefore of interest as potential cancer chemopreventive agents. In this study we assessed the effects of the phytocompounds garcinol and curcumin on histone and p53 modification in cancer cells, focussing on the breast tumour cell line MCF7. METHODS: Cell viability/proliferation assays, cell cycle analysis by flow cytometry, immunodetection of specific histone and p53 acetylation marks, western blotting, siRNA and RT-qPCR. RESULTS: Although treatment with curcumin, garcinol or the garcinol derivative LTK-14 hampered MCF7 cell proliferation, differential effects of these compounds on histone modifications were observed. Garcinol treatment resulted in a strong reduction in H3K18 acetylation, which is required for S phase progression. Similar effects of garcinol on H3K18 acetylation were observed in the osteosarcoma cells lines U2OS and SaOS2. In contrast, global levels of acetylated H4K16 and trimethylated H4K20 in MCF7 cells were elevated after garcinol treatment. This was accompanied by upregulation of DNA damage signalling markers such as γH2A.X, H3K56Ac, p53 and TIP60. In contrast, exposure of MCF7 cells to curcumin resulted in increased global levels of acetylated H3K18 and H4K16, and was less effective in inducing DNA damage markers. In addition to its effects on histone modifications, garcinol was found to block CBP/p300-mediated acetylation of the C-terminal activation domain of p53, but resulted in enhanced acetylation of p53K120, and accumulation of p53 in the cytoplasmic compartment. Finally, we show that the elevation of H4K20Me3 levels by garcinol correlated with increased expression of SUV420H2, and was prevented by siRNA targeting of SUV420H2. CONCLUSION: In summary, although garcinol and curcumin can both inhibit histone acetyltransferase activities, our results show that these compounds have differential effects on cancer cells in culture. Garcinol treatment alters expression of chromatin modifying enzymes in MCF7 cells, resulting in reprogramming of key histone and p53 PTMs and growth arrest, underscoring its potential as a cancer chemopreventive agent.

p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication.

The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3(') UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.

Apoferritin-Encapsulated Jerantinine A for Transferrin Receptor Targeting and Enhanced Selectivity in Breast Cancer Therapy.

The O-acetyl (or acetate) derivative of the Aspidosperma alkaloid Jerantinine A (JAa) elicits anti-tumor activity against cancer cell lines including mammary carcinoma cell lines irrespective of receptor status (0.14 < GI50 < 0.38 µM), targeting microtubule dynamics. By exploiting breast cancer cells' upregulated transferrin receptor 1 (TfR1) expression and apoferritin (AFt) recognition, we sought to develop an AFt JAa-delivery vehicle to enhance tumor-targeting and reduce systemic toxicity. Optimizing pH-mediated reassembly, ∼120 JAa molecules were entrapped within AFt. Western blot and flow cytometry demonstrate TfR1 expression in cancer cells. Enhanced internalization of 5-carboxyfluorescein-conjugated human AFt in SKBR3 and MDA-MB-231 cancer cells is observed compared to MRC5 fibroblasts. Accordingly, AFt-JAa delivers significantly greater intracellular JAa levels to SKBR3 and MDA-MB-231 cells than naked JAa (0.2 µM) treatment alone. Compared to naked JAa (0.2 µM), AFt-JAa achieves enhanced growth inhibition (2.5-14-fold; <0.02 µM < GI50 < 0.15 µM) in breast cancer cells; AFt-JAa treatment results in significantly reduced clonal survival, more profound cell cycle perturbation including G2/M arrest, greater reduction in cell numbers, and increased apoptosis compared to the naked agent (p < 0.01). Decreased PLK1 and Mcl-1 expression, together with the appearance of cleaved poly (ADP-ribose)-polymerase, corroborate the augmented potency of AFt-JAa. Hence, we demonstrate that AFt represents a biocompatible vehicle for targeted delivery of JAa, offering potential to minimize toxicity and enhance JAa activity in TfR1-expressing tumors.

PIP5K1α is Required for Promoting Tumor Progression in Castration-Resistant Prostate Cancer.

PIP5K1α has emerged as a promising drug target for the treatment of castration-resistant prostate cancer (CRPC), as it acts upstream of the PI3K/AKT signaling pathway to promote prostate cancer (PCa) growth, survival and invasion. However, little is known of the molecular actions of PIP5K1α in this process. Here, we show that siRNA-mediated knockdown of PIP5K1α and blockade of PIP5K1α action using its small molecule inhibitor ISA-2011B suppress growth and invasion of CRPC cells. We demonstrate that targeted deletion of the N-terminal domain of PIP5K1α in CRPC cells results in reduced growth and migratory ability of cancer cells. Further, the xenograft tumors lacking the N-terminal domain of PIP5K1α exhibited reduced tumor growth and aggressiveness in xenograft mice as compared to that of controls. The N-terminal domain of PIP5K1α is required for regulation of mRNA expression and protein stability of PIP5K1α. This suggests that the expression and oncogenic activity of PIP5K1α are in part dependent on its N-terminal domain. We further show that PIP5K1α acts as an upstream regulator of the androgen receptor (AR) and AR target genes including CDK1 and MMP9 that are key factors promoting growth, survival and invasion of PCa cells. ISA-2011B exhibited a significant inhibitory effect on AR target genes including CDK1 and MMP9 in CRPC cells with wild-type PIP5K1α and in CRPC cells lacking the N-terminal domain of PIP5K1α. These results indicate that the growth of PIP5K1α-dependent tumors is in part dependent on the integrity of the N-terminal sequence of this kinase. Our study identifies a novel functional mechanism involving PIP5K1α, confirming that PIP5K1α is an intriguing target for cancer treatment, especially for treatment of CRPC.

SUMOylation regulates the nuclear mobility of CREB binding protein and its association with nuclear bodies in live cells.

The lysine acetyltransferase CREB binding protein (CBP) is required for chromatin modification and transcription at many gene promoters. In fixed cells, a large proportion of CBP colocalises to PML or nuclear bodies. Using live cell imaging, we show here that YFP-tagged CBP expressed in HEK293 cells undergoes gradual accumulation in nuclear bodies, some of which are mobile and migrate towards the nuclear envelope. Deletion of a short lysine-rich domain that contains the major SUMO acceptor sites of CBP abrogated its ability to be SUMO modified, and prevented its association with endogenous SUMO-1/PML speckles in vivo. This SUMO-defective CBP showed enhanced ability to co-activate AML1-mediated transcription. Deletion mapping revealed that the SUMO-modified region was not sufficient for targeting CBP to PML bodies, as C-terminally truncated mutants containing this domain showed a strong reduction in accumulation at PML bodies. Fluorescence recovery after photo-bleaching (FRAP) experiments revealed that YFP-CBPDelta998-1087 had a retarded recovery time in the nucleus, as compared to YFP-CBP. These results indicate that SUMOylation regulates CBP function by influencing its shuttling between nuclear bodies and chromatin microenvironments.

Synthesis of micellar-like terpolymer nanoparticles with reductively-cleavable cross-links and evaluation of efficacy in 2D and 3D models of triple negative breast cancer.

Triple negative or basal-like breast cancer (TNBC) is characterised by aggressive progression, lack of standard therapies and poorer overall survival rates for patients. The bad prognosis, high rate of relapse and resistance against anticancer drugs have been associated with a highly abnormal loss of redox control in TNBC cells. Here, we developed docetaxel (DTX)-loaded micellar-like nanoparticles (MLNPs), designed to address the aberrant TNBC biology through the placement of redox responsive cross-links designed into a terpolymer. The MLNPs were derived from poly(ethyleneglycol)-b-poly(lactide)-co-poly(N3-α-ε-caprolactone) with a disulfide linker pendant from the caprolactone regions in order to cross-link adjacent chains. The terpolymer contained both polylactide and polycaprolactone to provide a balance of accessibility to reductive agents necessary to ensure stability in transit, but rapid micellar breakdown and concomitant drug release, when in breast cancer cells with increased levels of reducing agents. The empty MLNPs did not show any cytotoxicity in vitro in 2D monolayers of MDA-MB-231 (triple negative breast cancer), MCF7 (breast cancer) and MCF10A (normal breast epithelial cell line), whereas DTX-loaded reducible crosslinked MLNPs exhibited higher cytotoxicity against TNBC and breast cancer cells which present high intracellular levels of glutathione. Crosslinked and non-crosslinked MLNPs showed high and concentration-dependent cellular uptake in monolayers and tumour spheroids, including when assessed in co-cultures of TNBC cells and cancer-associated fibroblasts. DTX loaded crosslinked MLNPs showed the highest efficacy against 3D spheroids of TNBC, in addition the MLNPs also induced higher levels of apoptosis, as assessed by annexin V/PI assays and increased caspase 3/7 activity in MDA-MB-231 cells in comparison to cells treated with DTX-loaded un-crosslinked MLNP (used as a control) and free DTX. Taken together these data demonstrate that the terpolymer micellar-like nanoparticles with reducible crosslinks have high efficacy in both 2D and 3D in vitro cancer models by targeting the aberrant biology, i.e. loss of redox control of this type of tumour, thus may be promising and effective carrier systems for future clinical applications in TNBC.

Cardiac glycoside cerberin exerts anticancer activity through PI3K/AKT/mTOR signal transduction inhibition.

Natural products possess a significant role in anticancer therapy and many currently-used anticancer drugs are of natural origin. Cerberin (CR), a cardenolide isolated from the fruit kernel of Cerbera odollam, was found to potently inhibit cancer cell growth (GI50 values < 90 nM), colony formation and migration. Significant G2/M cell cycle arrest preceded time- and dose-dependent apoptosis-induction in human cancer cell lines corroborated by dose-and time-dependent PARP cleavage and caspase 3/7 activation, in addition to reduced Bcl-2 and Mcl-1 expression. CR potently inhibited PI3K/AKT/mTOR signalling depleting polo-like kinase 1 (PLK-1), c-Myc and STAT-3 expression. Additionally, CR significantly increased the generation of reactive oxygen species (ROS) producing DNA double strand breaks. Preliminary in silico biopharmaceutical assessment of CR predicted >60% bioavailability and rapid absorption; doses of 1-10 mg/kg CR were predicted to maintain efficacious unbound plasma concentrations (>GI50 value). CR's potent and selective anti-tumour activity, and its targeting of key signalling mechanisms pertinent to tumourigenesis support further preclinical evaluation of this cardiac glycoside.

MOZ-TIF2 inhibits transcription by nuclear receptors and p53 by impairment of CBP function.

Chromosomal rearrangements associated with acute myeloid leukemia (AML) include fusions of the genes encoding the acetyltransferase MOZ or MORF with genes encoding the nuclear receptor coactivator TIF2, p300, or CBP. Here we show that MOZ-TIF2 acts as a dominant inhibitor of the transcriptional activities of CBP-dependent activators such as nuclear receptors and p53. The dominant negative property of MOZ-TIF2 requires the CBP-binding domain (activation domain 1 [AD1]), and coimmunoprecipitation and fluorescent resonance energy transfer experiments show that MOZ-TIF2 interacts with CBP directly in vivo. The CBP-binding domain is also required for the ability of MOZ-TIF2 to extend the proliferative potential of murine bone marrow lineage-negative cells in vitro. We show that MOZ-TIF2 displays an aberrant nuclear distribution and that cells expressing this protein have reduced levels of cellular CBP, leading to depletion of CBP from PML bodies. In summary, our results indicate that disruption of the normal function of CBP and CBP-dependent activators is an important feature of MOZ-TIF2 action in AML.

Lipophilic activated ester prodrug approach for drug delivery to the intestinal lymphatic system.

The intestinal lymphatic system plays an important role in the pathophysiology of multiple diseases including lymphomas, cancer metastasis, autoimmune diseases, and human immunodeficiency virus (HIV) infection. It is thus an important compartment for delivery of drugs in order to treat diseases associated with the lymphatic system. Lipophilic prodrug approaches have been used in the past to take advantage of the intestinal lymphatic transport processes to deliver drugs to the intestinal lymphatics. Most of the approaches previously adopted were based on very bulky prodrug moieties such as those mimicking triglycerides (TG). We now report a study in which a lipophilic prodrug approach was used to efficiently deliver bexarotene (BEX) and retinoic acid (RA) to the intestinal lymphatic system using activated ester prodrugs. A range of carboxylic ester prodrugs of BEX were designed and synthesised and all of the esters showed improved association with chylomicrons, which indicated an improved potential for delivery to the intestinal lymphatic system. The conversion rate of the prodrugs to BEX was the main determinant in delivery of BEX to the intestinal lymphatics, and activated ester prodrugs were prepared to enhance the conversion rate. As a result, an 4-(hydroxymethyl)-1,3-dioxol-2-one ester prodrug of BEX was able to increase the exposure of the mesenteric lymph nodes (MLNs) to BEX 17-fold compared to when BEX itself was administered. The activated ester prodrug approach was also applied to another drug, RA, where the exposure of the MLNs was increased 2.4-fold through the application of a similar cyclic activated prodrug. Synergism between BEX and RA was also demonstrated in vitro by cell growth inhibition assays using lymphoma cell lines. In conclusion, the activated ester prodrug approach results in efficient delivery of drugs to the intestinal lymphatic system, which could benefit patients affected by a large number of pathological conditions.

Abnormal Clock Gene Expression and Locomotor Activity Rhythms in Two Month-Old Female APPSwe/PS1dE9 Mice.

BACKGROUND: In addition to cognitive decline, Alzheimer's Disease (AD) is also characterized by agitation and disruptions in activity and sleep. These symptoms typically occur in the evening or night and have been referred to as 'sundowning'. They are especially difficult for carers and there are no specific drug treatments. There is increasing evidence that these symptoms reflect pathology of circadian rhythm generation and transmission. OBJECTIVE: We investigated whether a transgenic mouse model relevant to AD (APPswe/PS1dE9) exhibits circadian alterations in locomotor activity in their home cage and whether expression of clock genes involved in the regulation of the circadian cycle is abnormal in the hippocampus and medulla-pons brain regions isolated from these mice. RESULTS: In 2month old female mice the APPswe/PS1dE9 transgene alters levels and patterns in circadian rhythm of locomotor activity. Expression of the clock genes Per1, Per2, Cry1 and Cry2 was found to increase at night compared to day in wild-type control mice in the medulla/pons. This effect was blunted for Cry1 and Cry2 gene expression in APPswe/PS1dE9. CONCLUSION: This study suggests altered circadian regulation of locomotor activity is abnormal in female APPswe/ PS1dE9 mice and that this alteration has biomolecular analogies in a widely available model of AD. The early age at which these effects are manifest suggests that these circadian effects may precede plaque development. The APPswe/PS1dE9 mouse genetic model may have potential to serve as a tool in understanding the neuropathology of circadian abnormalities in AD and as a model system to test novel therapeutic agents for these symptoms.

Heterodimers of photoreceptor-specific nuclear receptor (PNR/NR2E3) and peroxisome proliferator-activated receptor-γ (PPARγ) are disrupted by retinal disease-associated mutations.

Photoreceptor-specific nuclear receptor (PNR/NR2E3) and Tailless homolog (TLX/NR2E1) are human orthologs of the NR2E group, a subgroup of phylogenetically related members of the nuclear receptor (NR) superfamily of transcription factors. We assessed the ability of these NRs to form heterodimers with other members of the human NRs representing all major subgroups. The TLX ligand-binding domain (LBD) did not appear to form homodimers or interact directly with any other NR tested. The PNR LBD was able to form homodimers, but also exhibited robust interactions with the LBDs of peroxisome proliferator-activated receptor-γ (PPARγ)/NR1C3 and thyroid hormone receptor b (TRb) TRß/NR1A2. The binding of PNR to PPARγ was specific for this paralog, as no interaction was observed with the LBDs of PPARα/NR1C1 or PPARδ/NR1C2. In support of these findings, PPARγ and PNR were found to be co-expressed in human retinal tissue extracts and could be co-immunoprecipitated as a native complex. Selected sequence variants in the PNR LBD associated with human retinopathies, or a mutation in the dimerization region of PPARγ LBD associated with familial partial lipodystrophy type 3, were found to disrupt PNR/PPARγ complex formation. Wild-type PNR, but not a PNR309G mutant, was able to repress PPARγ-mediated transcription in reporter assays. In summary, our results reveal novel heterodimer interactions in the NR superfamily, suggesting previously unknown functional interactions of PNR with PPARγ and TRß that have potential importance in retinal development and disease.

MOZ fusion proteins in acute myeloid leukaemia.

MOZ (monocytic leukaemia zinc finger protein; also known as ZNF220 or MYST3) is a member of the MYST family of protein acetyltransferases. Chromosomal translocations involving the MOZ gene are associated with AML (acute myeloid leukaemia), suggesting that it has a role in haematopoiesis. Recurrent reciprocal translocations fuse the MOZ gene [or the gene encoding MORF (MOZ-related factor); also known as MYST4] to genes encoding the nuclear receptor co-activators CBP [CREB (cAMP response element-binding protein)-binding protein], p300 or the p160 protein TIF2 (transcription intermediary factor 2). The resulting fusion proteins can transform haematopoietic progenitors in vitro, and induce myeloproliferative disease in mice. Recent insights into the molecular mechanisms underlying these effects indicate that MOZ fusion proteins interfere with the activities of transcription factors such as nuclear receptors, p53 and Runx proteins. Our studies suggest that subverting the function of cellular CBP and p300 proteins may play a key role in this process. Here we review the recent progress in understanding the role of MOZ fusion proteins in the aetiology of AML.

PRAME is a golgi-targeted protein that associates with the Elongin BC complex and is upregulated by interferon-gamma and bacterial PAMPs.

Preferentially expressed antigen in melanoma (PRAME) has been described as a cancer-testis antigen and is associated with leukaemias and solid tumours. Here we show that PRAME gene transcription in leukaemic cell lines is rapidly induced by exposure of cells to bacterial PAMPs (pathogen associated molecular patterns) in combination with type 2 interferon (IFNγ). Treatment of HL60 cells with lipopolysaccharide or peptidoglycan in combination with IFNγ resulted in a rapid and transient induction of PRAME transcription, and increased association of PRAME transcripts with polysomes. Moreover, treatment with PAMPs/IFNγ also modulated the subcellular localisation of PRAME proteins in HL60 and U937 cells, resulting in targeting of cytoplasmic PRAME to the Golgi. Affinity purification studies revealed that PRAME associates with Elongin B and Elongin C, components of Cullin E3 ubiquitin ligase complexes. This occurs via direct interaction of PRAME with Elongin C, and PRAME colocalises with Elongins in the Golgi after PAMP/IFNγ treatment. PRAME was also found to co-immunoprecipitate core histones, consistent with its partial localisation to the nucleus, and was found to bind directly to histone H3 in vitro. Thus, PRAME is upregulated by signalling pathways that are activated in response to infection/inflammation, and its product may have dual functions as a histone-binding protein, and in directing ubiquitylation of target proteins for processing in the Golgi.

Global histone modifications in breast cancer correlate with tumor phenotypes, prognostic factors, and patient outcome.

Post-translational histone modifications are known to be altered in cancer cells, and loss of selected histone acetylation and methylation marks has recently been shown to predict patient outcome in human carcinoma. Immunohistochemistry was used to detect a series of histone lysine acetylation (H3K9ac, H3K18ac, H4K12ac, and H4K16ac), lysine methylation (H3K4me2 and H4K20me3), and arginine methylation (H4R3me2) marks in a well-characterized series of human breast carcinomas (n = 880). Tissue staining intensities were assessed using blinded semiquantitative scoring. Validation studies were done using immunofluorescence staining and Western blotting. Our analyses revealed low or absent H4K16ac in the majority of breast cancer cases (78.9%), suggesting that this alteration may represent an early sign of breast cancer. There was a highly significant correlation between histone modifications status, tumor biomarker phenotype, and clinical outcome, where high relative levels of global histone acetylation and methylation were associated with a favorable prognosis and detected almost exclusively in luminal-like breast tumors (93%). Moderate to low levels of lysine acetylation (H3K9ac, H3K18ac, and H4K12ac), lysine (H3K4me2 and H4K20me3), and arginine methylation (H4R3me2) were observed in carcinomas of poorer prognostic subtypes, including basal carcinomas and HER-2-positive tumors. Clustering analysis identified three groups of histone displaying distinct pattern in breast cancer, which have distinct relationships to known prognostic factors and clinical outcome. This study identifies the presence of variations in global levels of histone marks in different grades, morphologic types, and phenotype classes of invasive breast cancer and shows that these differences have clinical significance.