A highly selective purine-based inhibitor of CSF1R potently inhibits osteoclast differentiation.

The colony-stimulating factor 1 receptor (CSF1R) plays an important role in the regulation of many inflammatory processes, and overexpression of the kinase is implicated in several disease states. Identifying selective, small-molecule inhibitors of CSF1R may be a crucial step toward treating these disorders. Through modelling, synthesis, and a systematic structure-activity relationship study, we have identified a number of potent and highly selective purine-based inhibitors of CSF1R. The optimized 6,8-disubstituted antagonist, compound 9, has enzymatic IC50 of 0.2 nM, and displays a strong affinity toward the autoinhibited form of CSF1R, contrasting that of other previously reported inhibitors. As a result of its binding mode, the inhibitor shows excellent selectivity (Selectivity score: 0.06), evidenced by profiling towards a panel of 468 kinases. In cell-based assays, this inhibitor shows dose-dependent blockade of CSF1-mediated downstream signalling in murine bone marrow-derived macrophages (IC50 = 106 nM) as well as disruption of osteoclast differentiation at nanomolar levels. In vivo experiments, however, indicate that improve metabolic stability is needed in order to further progress this compound class.

Small-molecule inhibitors of human mitochondrial DNA transcription.

Altered expression of mitochondrial DNA (mtDNA) occurs in ageing and a range of human pathologies (for example, inborn errors of metabolism, neurodegeneration and cancer). Here we describe first-in-class specific inhibitors of mitochondrial transcription (IMTs) that target the human mitochondrial RNA polymerase (POLRMT), which is essential for biogenesis of the oxidative phosphorylation (OXPHOS) system1-6. The IMTs efficiently impair mtDNA transcription in a reconstituted recombinant system and cause a dose-dependent inhibition of mtDNA expression and OXPHOS in cell lines. To verify the cellular target, we performed exome sequencing of mutagenized cells and identified a cluster of amino acid substitutions in POLRMT that cause resistance to IMTs. We obtained a cryo-electron microscopy (cryo-EM) structure of POLRMT bound to an IMT, which further defined the allosteric binding site near the active centre cleft of POLRMT. The growth of cancer cells and the persistence of therapy-resistant cancer stem cells has previously been reported to depend on OXPHOS7-17, and we therefore investigated whether IMTs have anti-tumour effects. Four weeks of oral treatment with an IMT is well-tolerated in mice and does not cause OXPHOS dysfunction or toxicity in normal tissues, despite inducing a strong anti-tumour response in xenografts of human cancer cells. In summary, IMTs provide a potent and specific chemical biology tool to study the role of mtDNA expression in physiology and disease.

Identification and Characterization of Dual Inhibitors of the USP25/28 Deubiquitinating Enzyme Subfamily.

The ubiquitin proteasome system is widely postulated to be a new and important field of drug discovery for the future, with the ubiquitin specific proteases (USPs) representing one of the more attractive target classes within the area. Many USPs have been linked to critical axes for therapeutic intervention, and the finding that USP28 is required for c-Myc stability suggests that USP28 inhibition may represent a novel approach to targeting this so far undruggable oncogene. Here, we describe the discovery of the first reported inhibitors of USP28, which we demonstrate are able to bind to and inhibit USP28, and while displaying a dual activity against the closest homologue USP25, these inhibitors show a high degree of selectivity over other deubiquitinases (DUBs). The utility of these compounds as valuable probes to investigate and further explore cellular DUB biology is highlighted by the demonstration of target engagement against both USP25 and USP28 in cells. Furthermore, we demonstrate that these inhibitors are able to elicit modulation of both the total levels and the half-life of the c-Myc oncoprotein in cells and also induce apoptosis and loss of cell viability in a range of cancer cell lines. We however observed a narrow therapeutic index compared to a panel of tissue-matched normal cell lines. Thus, it is hoped that these probes and data presented herein will further advance our understanding of the biology and tractability of DUBs as potential future therapeutic targets.

ATM Kinase Inhibitors: HTS Cellular Imaging Assay Using Cellomics™ ArrayScan VTI Platform.

Small molecule inhibitors of the ATM pathway could represent a promising opportunity for cancer therapy, working either by enhancing the clinical efficacy of radiotherapy and existing chemotherapies or by synthetic lethality-based mechanisms. In this chapter, we describe a high-throughput, high-content imaging assay monitoring levels of ATM phosphorylation at Serine 1981 following induction of DNA damage by ionizing radiation.

Nuclear respiratory factor 1 and endurance exercise promote human telomere transcription.

DNA breaks activate the DNA damage response and, if left unrepaired, trigger cellular senescence. Telomeres are specialized nucleoprotein structures that protect chromosome ends from persistent DNA damage response activation. Whether protection can be enhanced to counteract the age-dependent decline in telomere integrity is a challenging question. Telomeric repeat-containing RNA (TERRA), which is transcribed from telomeres, emerged as important player in telomere integrity. However, how human telomere transcription is regulated is still largely unknown. We identify nuclear respiratory factor 1 and peroxisome proliferator-activated receptor γ coactivator 1α as regulators of human telomere transcription. In agreement with an upstream regulation of these factors by adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), pharmacological activation of AMPK in cancer cell lines or in normal nonproliferating myotubes up-regulated TERRA, thereby linking metabolism to telomere fitness. Cycling endurance exercise, which is associated with AMPK activation, increased TERRA levels in skeletal muscle biopsies obtained from 10 healthy young volunteers. The data support the idea that exercise may protect against aging.

Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9.

Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 µM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM).

Whole transcriptome profiling of patient-derived xenograft models as a tool to identify both tumor and stromal specific biomarkers.

The tumor microenvironment is emerging as a key regulator of cancer growth and progression, however the exact mechanisms of interaction with the tumor are poorly understood. Whilst the majority of genomic profiling efforts thus far have focused on the tumor, here we investigate RNA-Seq as a hypothesis-free tool to generate independent tumor and stromal biomarkers, and explore tumor-stroma interactions by exploiting the human-murine compartment specificity of patient-derived xenografts (PDX).Across a pan-cancer cohort of 79 PDX models, we determine that mouse stroma can be separated into distinct clusters, each corresponding to a specific stromal cell type. This implies heterogeneous recruitment of mouse stroma to the xenograft independent of tumor type. We then generate cross-species expression networks to recapitulate a known association between tumor epithelial cells and fibroblast activation, and propose a potentially novel relationship between two hypoxia-associated genes, human MIF and mouse Ddx6. Assessment of disease subtype also reveals MMP12 as a putative stromal marker of triple-negative breast cancer. Finally, we establish that our ability to dissect recruited stroma from trans-differentiated tumor cells is crucial to identifying stem-like poor-prognosis signatures in the tumor compartment.In conclusion, RNA-Seq is a powerful, cost-effective solution to global analysis of human tumor and mouse stroma simultaneously, providing new insights into mouse stromal heterogeneity and compartment-specific disease markers that are otherwise overlooked by alternative technologies. The study represents the first comprehensive analysis of its kind across multiple PDX models, and supports adoption of the approach in pre-clinical drug efficacy studies, and compartment-specific biomarker discovery.

Polycomb repressive complex 2 and H3K27me3 cooperate with H3K9 methylation to maintain heterochromatin protein 1α at chromatin.

Methylation of histone H3 on lysine 9 or 27 is crucial for heterochromatin formation. Previously considered hallmarks of, respectively, constitutive and facultative heterochromatin, recent evidence has accumulated in favor of coexistence of these two marks and their cooperation in gene silencing maintenance. H3K9me2/3 ensures anchorage at chromatin of heterochromatin protein 1α (HP1α), a main component of heterochromatin. HP1α chromoshadow domain, involved in dimerization and interaction with partners, has additional but still unclear roles in HP1α recruitment to chromatin. Because of previously suggested links between polycomb repressive complex 2 (PRC2), which catalyzes H3K27 methylation, and HP1α, we tested whether PRC2 may regulate HP1α abundance at chromatin. We found that the EZH2 and SUZ12 subunits of PRC2 are required for HP1α stability, as knockdown of either protein led to HP1α degradation. Similar results were obtained upon overexpression of H3K27me2/3 demethylases. We further showed that binding of HP1α/ß/γ to H3K9me3 peptides is greatly increased in the presence of H3K27me3, and this is dependent on PRC2. These data fit with recent proteomic studies identifying PRC2 as an indirect H3K9me3 binder in mouse tissues and suggest the existence of a cooperative mechanism of HP1α anchorage at chromatin involving H3 methylation on both K9 and K27 residues.

Development of a high-content high-throughput screening assay for the discovery of ATM signaling inhibitors.

The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.

Notch protection against apoptosis in T-ALL cells mediated by GIMAP5.

Recent studies have highlighted the role of Notch signalling in the development of T cell acute lymphoblasic leukaemia (T-ALL). Over-expression of Notch3 and gain of function mutations in the Notch1 gene have been reported. The aims of this study were to determine the effect of Notch signalling on apoptosis in human T-ALL cell lines and to identify targets of Notch signalling that may mediate this effect. Functional studies showed that inhibition of Notch signalling using gamma secretase inhibitors promoted glucocorticoid-induced apoptosis in cells carrying gain of function mutations in Notch1. Moreover, ectopic expression of constitutively activated Notch provided protection against glucocorticoid-induced apoptosis, indicating that signalling via Notch may also contribute to the development of T-ALL by conferring resistance to apoptosis. Microarray analysis revealed that GIMAP5, a gene coding for an anti-apoptotic intracellular protein, is upregulated by Notch in T-ALL cell lines. Knockdown of GIMAP5 expression using siRNA promoted glucocorticoid-induced apoptosis in T-ALL cells carrying gain of function mutations in Notch1 and in T-ALL cells engineered to express ectopic constitutively activated Notch indicating that Notch signalling protects T-ALL cells from apoptosis by upregulating the expression of GIMAP5.

Overlapping promoter targeting by Elk-1 and other divergent ETS-domain transcription factor family members.

ETS-domain transcription factors play important roles in controlling gene expression in a variety of different contexts; however, these proteins bind to very similar sites and it is unclear how in vivo specificity is achieved. In silico analysis is unlikely to reveal specific targets for individual family members and direct experimental approaches are therefore required. Here, we take advantage of an inducible dominant-negative expression system to identify a group of novel target genes for the ETS-domain transcription factor Elk-1. Elk-1 is thought to mainly function through cooperation with a second transcription factor SRF, but the targets we identify are largely SRF-independent. Furthermore, we demonstrate that there is a high degree of overlapping, cell type-specific, target gene binding by Elk-1 and other ETS-domain transcription factors. Our results are therefore consistent with the notion that there is a high degree of functional redundancy in target gene regulation by ETS-domain transcription factors in addition to the specific target gene regulation that can be dictated through heterotypic interactions exemplified by the Elk-1-SRF complex.

Elucidation of the ELK1 target gene network reveals a role in the coordinate regulation of core components of the gene regulation machinery.

Transcription factors play an important role in orchestrating the activation of specific networks of genes through targeting their proximal promoter and distal enhancer regions. However, it is unclear how the specificity of downstream responses is maintained by individual members of transcription-factor families and, in most cases, what their target repertoire is. We have used ChIP-chip analysis to identify the target genes of the ETS-domain transcription factor ELK1. Two distinct modes of ELK1 target gene selection are identified; the first involves redundant promoter binding with other ETS-domain family members; the second occurs through combinatorial binding with a second transcription factor SRF, which specifies a unique group of target genes. One of the most prominent groups of genes forming the ELK1 target network includes classes involved in core gene expression control, namely, components of the basal transcriptional machinery, the spliceosome and the ribosome. Amongst the set of genes encoding the basal transcription machinery components, are a functionally linked subset of GTFs and TAFs. Our study, therefore, reveals an unsuspected level of coordinate regulation of components of the core gene expression control machinery and also identifies two different modes of promoter targeting through binding with a second transcription factor or redundant binding with other ETS-domain family members.

The 5' region of the human hSUV3 gene encoding mitochondrial DNA and RNA helicase: promoter characterization and alternative pre-mRNA splicing.

The human nuclear hSUV3 gene encodes ATP-dependent RNA and DNA helicase, which predominantly localizes in the mitochondria. In yeast, the Suv3 helicase is a component of mitochondrial degradosome, a two-subunit complex, which degrades aberrant mtRNAs. In contrast to the well-documented physiological role of the yeast SUV3, the function of its human orthologue remains unknown. In this report, we have analyzed the hSUV3 5' genomic region. Our data suggest that hSUV3 is a housekeeping gene. Deletion analysis and in vitro mutagenesis revealed the presence of an enhancer region and regulatory elements in basal promoter including: (i) direct 10-bp-long repeats, which share significant sequence similarity with the consensus for the NF-kappaB/Rel family transcription factors, (ii) Sp1 general transcription factor binding site, and (iii) NRF-1 transcription factor binding sites, the latter typical for nuclear-encoded mitochondrial genes. Furthermore, we show that the 5' region of the hSUV3 pre-mRNA can be alternatively spliced.

Hsp90 chaperones wild-type p53 tumor suppressor protein.

Immortalized human fibroblasts were used to investigate the putative interactions of the Hsp90 molecular chaperone with the wild-type p53 tumor suppressor protein. We show that geldanamycin or radicicol, specific inhibitors of Hsp90, diminish specific wild-type p53 binding to the p21 promoter sequence. Consequently, these inhibitors decrease p21 mRNA levels, which lead to a reduction in cellular p21/Waf1 protein, known to induce cell cycle arrest. In control experiments, we show that neither geldanamycin nor radicicol affect p53 mRNA levels. A minor decrease in p53 protein level following the treatment of human fibroblasts with the inhibitors suggests the potential involvement of Hsp90 in the stabilization of wild-type p53. To support our in vivo findings, we used a reconstituted system with highly purified recombinant proteins to examine the effects of Hsp90 on wild-type p53 binding to the p21 promoter sequence. The human recombinant Hsp90 alpha-isoform as well as bovine brain Hsp90 were purified to homogeneity. Both of these molecular chaperones displayed ATPase activity and the ability to refold heat-inactivated luciferase in a geldanamycin- and radicicol-sensitive manner, suggesting that post-translational modifications are not involved in the modulation of Hsp90alpha activity. We show that the incubation of recombinant p53 at 37 degrees C decreases the level of its wild-type conformation and strongly inhibits the in vitro binding of p53 to the p21 promoter sequence. Interestingly, Hsp90 in an ATP-dependent manner can positively modulate p53 DNA binding after incubation at physiological temperature of 37 degrees C. Other recombinant human chaperones from Hsp70 and Hsp40 families were not able to efficiently substitute Hsp90 in this reaction. Consistent with our in vivo results, geldanamycin can suppress Hsp90 ability to regulate in vitro p53 DNA binding to the promoter sequence. In summary, the results presented in this article state that chaperone activity of Hsp90 is important for the transcriptional activity of genotypically wild-type p53.

Cell cycle-regulated transcription through the FHA domain of Fkh2p and the coactivator Ndd1p.

Recent studies in Saccharomyces cerevisiae by using global approaches have significantly enhanced our knowledge of the components involved in the transcriptional regulation of the cell cycle. The Mcm1p-Fkh2p complex, in combination with the coactivator Ndd1p, plays an important role in the cell cycle-dependent expression of the CLB2 gene cluster during the G2 and M phases ([4-7]; see [8-10]for reviews). Fkh2p is phosphorylated in a cell cycle-dependent manner, and peak phosphorylation occurs coincidentally with maximal expression of Mcm1p-Fkh2p-dependent gene expression. However, the mechanism by which this complex is activated in a cell cycle-dependent manner is unknown. Here, we demonstrate that the forkhead-associated (FHA) domain of Fkh2p directs cell cycle-regulated transcription and that the activity of this domain is dependent on the coactivator Ndd1p. Ndd1p was found to be phosphorylated in a cell cycle-dependent manner by Cdc28p-Clb2p, and, importantly, this phosphorylation event promotes interactions between Ndd1p and the FHA domain of Fkh2p. Furthermore, mutation of the FHA domain blocks these phosphorylation-dependent interactions and abolishes transcriptional activity. Our data therefore link the transcriptional activity of the FHA domain with cell cycle-dependent phosphorylation of the coactivator Ndd1p and reveal a mechanism that permits precise temporal activation of the Mcm1p-Fkh2p complex.

Molecular determinants of the cell-cycle regulated Mcm1p-Fkh2p transcription factor complex.

The MADS-box transcription factor Mcm1p and forkhead (FKH) transcription factor Fkh2p act in a DNA-bound complex to regulate cell-cycle dependent expression of the CLB2 cluster in Saccharomyces cerevisiae. Binding of Fkh2p requires prior binding by Mcm1p. Here we have investigated the molecular determinants governing the formation of the Mcm1p- Fkh2p complex. Fkh2p exhibits cooperativity in complex formation with Mcm1p and we have mapped a small region of Fkh2p located immediately upstream of the FKH DNA binding domain that is required for this cooperativity. This region is lacking in the related protein Fkh1p that cannot form ternary complexes with Mcm1p. A second region is identified that inhibits Mcm1p-independent DNA binding by Fkh2p. The spacing between the Mcm1p and Fkh2p binding sites is also a critical determinant for complex formation. We also show that Fkh2p can form ternary complexes with the human counterpart of Mcm1p, serum response factor (SRF). Mutations at analogous positions in Mcm1p, which are known to affect SRF interaction with its partner protein Elk-1, abrogate complex formation with Fkh2p, demonstrating evolutionary conservation of coregulatory protein binding surfaces. Our data therefore provide molecular insights into the mechanisms of Mcm1p- Fkh2p complex formation and more generally aid our understanding of MADS-box protein function.