The molecular chaperone heat shock protein 90 (HSP90) works in concert with co-chaperones to stabilize its client proteins, which include multiple drivers of oncogenesis and malignant progression. Pharmacologic inhibitors of HSP90 have been observed to exert a wide range of effects on the proteome, including depletion of client proteins, induction of heat shock proteins, dissociation of co-chaperones from HSP90, disruption of client protein signaling networks, and recruitment of the protein ubiquitylation and degradation machinery-suggesting widespread remodeling of cellular protein complexes. However, proteomics studies to date have focused on inhibitor-induced changes in total protein levels, often overlooking protein complex alterations. Here, we use size-exclusion chromatography in combination with mass spectrometry (SEC-MS) to characterize the early changes in native protein complexes following treatment with the HSP90 inhibitor tanespimycin (17-AAG) for 8 h in the HT29 colon adenocarcinoma cell line. After confirming the signature cellular response to HSP90 inhibition (e.g., induction of heat shock proteins, decreased total levels of client proteins), we were surprised to find only modest perturbations to the global distribution of protein elution profiles in inhibitor-treated HT29 cells at this relatively early time-point. Similarly, co-chaperones that co-eluted with HSP90 displayed no clear difference between control and treated conditions. However, two distinct analysis strategies identified multiple inhibitor-induced changes, including known and unknown components of the HSP90-dependent proteome. We validate two of these-the actin-binding protein Anillin and the mitochondrial isocitrate dehydrogenase 3 complex-as novel HSP90 inhibitor-modulated proteins. We present this dataset as a resource for the HSP90, proteostasis, and cancer communities (https://www.bioinformatics.babraham.ac.uk/shiny/HSP90/SEC-MS/), laying the groundwork for future mechanistic and therapeutic studies related to HSP90 pharmacology. Data are available via ProteomeXchange with identifier PXD033459..

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies..

Most small molecules interact with several target proteins but this polypharmacology is seldom comprehensively investigated or explicitly exploited during drug discovery. Here, we use computational and experimental methods to identify and systematically characterize the kinase cross-pharmacology of representative HSP90 inhibitors. We demonstrate that the resorcinol clinical candidates ganetespib and, to a lesser extent, luminespib, display unique off-target kinase pharmacology as compared with other HSP90 inhibitors. We also demonstrate that polypharmacology evolved during the optimization to discover luminespib and that the hit, leads, and clinical candidate all have different polypharmacological profiles. We therefore recommend the computational and experimental characterization of polypharmacology earlier in drug discovery projects to unlock new multi-target drug design opportunities..

The undruggable nature of oncogenic Myc transcription factors poses a therapeutic challenge in neuroblastoma, a pediatric cancer in which MYCN amplification is strongly associated with unfavorable outcome. Here, we show that CYC065 (fadraciclib), a clinical inhibitor of CDK9 and CDK2, selectively targeted MYCN-amplified neuroblastoma via multiple mechanisms. CDK9 - a component of the transcription elongation complex P-TEFb - bound to the MYCN-amplicon superenhancer, and its inhibition resulted in selective loss of nascent MYCN transcription. MYCN loss led to growth arrest, sensitizing cells for apoptosis following CDK2 inhibition. In MYCN-amplified neuroblastoma, MYCN invaded active enhancers, driving a transcriptionally encoded adrenergic gene expression program that was selectively reversed by CYC065. MYCN overexpression in mesenchymal neuroblastoma was sufficient to induce adrenergic identity and sensitize cells to CYC065. CYC065, used together with temozolomide, a reference therapy for relapsed neuroblastoma, caused long-term suppression of neuroblastoma growth in vivo, highlighting the clinical potential of CDK9/2 inhibition in the treatment of MYCN-amplified neuroblastoma..

KRAS mutation is a key driver of pancreatic cancer and PI3K pathway activity is an additional requirement for Kras-induced tumorigenesis. Clinical trials of PI3K pathway inhibitors in pancreatic cancer have shown limited responses. Understanding the molecular basis for this lack of efficacy may direct future treatment strategies with emerging PI3K inhibitors. We sought new therapeutic approaches that synergize with PI3K inhibitors through pooled CRISPR modifier genetic screening and a drug combination screen. ERBB family receptor tyrosine kinase signaling and mTOR signaling were key modifiers of sensitivity to alpelisib and pictilisib. Inhibition of the ERBB family or mTOR was synergistic with PI3K inhibition in spheroid, stromal cocultures. Near-complete loss of ribosomal S6 phosphorylation was associated with synergy. Genetic alterations in the ERBB-PI3K signaling axis were associated with decreased survival of patients with pancreatic cancer. Suppression of the PI3K/mTOR axis is potentiated by dual PI3K and ERBB family or mTOR inhibition. Surprisingly, despite the presence of oncogenic KRAS, thought to bestow independence from receptor tyrosine kinase signaling, inhibition of the ERBB family blocks downstream pathway activation and synergizes with PI3K inhibitors. Further exploration of these therapeutic combinations is warranted for the treatment of pancreatic cancer..

BACKGROUND AND AIMS: Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy. APPROACH AND RESULTS: High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA-sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide-treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild-type models showed only stable disease over treatment. CONCLUSIONS: MIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics..

Checkpoint kinase 1 (CHK1) is a key mediator of the DNA damage response that regulates cell-cycle progression, DNA damage repair, and DNA replication. Small-molecule CHK1 inhibitors sensitize cancer cells to genotoxic agents and have shown single-agent preclinical activity in cancers with high levels of replication stress. However, the underlying genetic determinants of CHK1 inhibitor sensitivity remain unclear. We used the developmental clinical drug SRA737 in an unbiased large-scale siRNA screen to identify novel mediators of CHK1 inhibitor sensitivity and uncover potential combination therapies and biomarkers for patient selection. We identified subunits of the B-family of DNA polymerases (POLA1, POLE, and POLE2) whose silencing sensitized the human A549 non-small cell lung cancer (NSCLC) and SW620 colorectal cancer cell lines to SRA737. B-family polymerases were validated using multiple siRNAs in a panel of NSCLC and colorectal cancer cell lines. Replication stress, DNA damage, and apoptosis were increased in human cancer cells following depletion of the B-family DNA polymerases combined with SRA737 treatment. Moreover, pharmacologic blockade of B-family DNA polymerases using aphidicolin or CD437 combined with CHK1 inhibitors led to synergistic inhibition of cancer cell proliferation. Furthermore, low levels of POLA1, POLE, and POLE2 protein expression in NSCLC and colorectal cancer cells correlated with single-agent CHK1 inhibitor sensitivity and may constitute biomarkers of this phenotype. These findings provide a potential basis for combining CHK1 and B-family polymerase inhibitors in cancer therapy. SIGNIFICANCE: These findings demonstrate how the therapeutic benefit of CHK1 inhibitors may potentially be enhanced and could have implications for patient selection and future development of new combination therapies..

Polypharmacology plays an important role in defining response and adverse effects of drugs. For some mechanisms, experimentally mapping polypharmacology is commonplace, although this is typically done within the same protein class. Four PARP inhibitors have been approved by the FDA as cancer therapeutics, yet a precise mechanistic rationale to guide clinicians on which to choose for a particular patient is lacking. The four drugs have largely similar PARP family inhibition profiles, but several differences at the molecular and clinical level have been reported that remain poorly understood. Here, we report the first comprehensive characterization of the off-target kinase landscape of four FDA-approved PARP drugs. We demonstrate that all four PARP inhibitors have a unique polypharmacological profile across the kinome. Niraparib and rucaparib inhibit DYRK1s, CDK16 and PIM3 at clinically achievable, submicromolar concentrations. These kinases represent the most potently inhibited off-targets of PARP inhibitors identified to date and should be investigated further to clarify their potential implications for efficacy and safety in the clinic. Moreover, broad kinome profiling is recommended for the development of PARP inhibitors as PARP-kinase polypharmacology could potentially be exploited to modulate efficacy and side-effect profiles..

Receptor tyrosine kinases (RTKs) are often overexpressed or mutated in cancers and drive tumor growth and metastasis. In the current model of RTK signaling, including that of MET, downstream phosphatidylinositol 3-kinase (PI3K) mediates both cell proliferation and cell migration, whereas the small guanosine triphosphatase (GTPase) Rac1 mediates cell migration. However, in cultured NIH3T3 and glioblastoma cells, we found that class I PI3K mediated oncogenic MET-induced cell migration but not anchorage-independent growth. In contrast, Rac1 regulated both processes in distinct ways. Downstream of PI3K, Rac1 mediated cell migration through its GTPase activity, whereas independently of PI3K, Rac1 mediated anchorage-independent growth in a GTPase-independent manner through an adaptor function. Through its RKR motif, Rac1 formed a complex with the kinase mTOR to promote its translocation to the plasma membrane, where its activity promoted anchorage-independent growth of the cell cultures. Inhibiting mTOR with rapamycin suppressed the growth of subcutaneous MET-mutant cell grafts in mice, including that of MET inhibitor-resistant cells. These findings reveal a GTPase-independent role for Rac1 in mediating a PI3K-independent MET-to-mTOR pathway and suggest alternative or combined strategies that might overcome resistance to RTK inhibitors in patients with cancer..

Cyclin-dependent kinases (CDKs) contribute to the cancer hallmarks of uncontrolled proliferation and increased survival. As a result, over the last two decades substantial efforts have been directed towards identification and development of pharmaceutical CDK inhibitors. Insights into the biological consequences of CDK inhibition in specific tumor types have led to the successful development of CDK4/6 inhibitors as treatments for certain types of breast cancer. More recently, a new generation of pharmaceutical inhibitors of CDK enzymes that regulate the transcription of key oncogenic and pro-survival proteins, including CDK9, have entered clinical development. Here, we provide the first disclosure of the chemical structure of fadraciclib (CYC065), a CDK inhibitor and clinical candidate designed by further optimization from the aminopurine scaffold of seliciclib. We describe its synthesis and mechanistic characterization. Fadraciclib exhibits improved potency and selectivity for CDK2 and CDK9 compared to seliciclib, and also displays high selectivity across the kinome. We show that the mechanism of action of fadraciclib is consistent with potent inhibition of CDK9-mediated transcription, decreasing levels of RNA polymerase II C-terminal domain serine 2 phosphorylation, the pro-survival protein Myeloid Cell Leukemia 1 (MCL1) and MYC oncoprotein, and inducing rapid apoptosis in cancer cells. This cellular potency and mechanism of action translate to promising anti-cancer activity in human leukemia mouse xenograft models. Studies of leukemia cell line sensitivity identify mixed lineage leukemia (MLL) gene status and the level of B-cell lymphoma 2 (BCL2) family proteins as potential markers for selection of patients with greater sensitivity to fadraciclib. We show that the combination of fadraciclib with BCL2 inhibitors, including venetoclax, is synergistic in leukemic cell models, as predicted from simultaneous inhibition of MCL1 and BCL2 pro-survival pathways. Fadraciclib preclinical pharmacology data support its therapeutic potential in CDK9- or CDK2-dependent cancers and as a rational combination with BCL2 inhibitors in hematological malignancies. Fadraciclib is currently in Phase 1 clinical studies in patients with advanced solid tumors (NCT02552953) and also in combination with venetoclax in patients with relapsed or refractory chronic lymphocytic leukemia (CLL) (NCT03739554) and relapsed refractory acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) (NCT04017546)..

The Mediator is an evolutionarily conserved, multi-subunit complex that regulates multiple steps of transcription. Mediator activity is regulated by the reversible association of a four-subunit module comprising CDK8 or CDK19 kinases, together with cyclin C, MED12 or MED12L, and MED13 or MED13L. Mutations in MED12, MED13, and MED13L were previously identified in syndromic developmental disorders with overlapping phenotypes. Here, we report CDK8 mutations (located at 13q12.13) that cause a phenotypically related disorder. Using whole-exome or whole-genome sequencing, and by international collaboration, we identified eight different heterozygous missense CDK8 substitutions, including 10 shown to have arisen de novo, in 12 unrelated subjects; a recurrent mutation, c.185C>T (p.Ser62Leu), was present in five individuals. All predicted substitutions localize to the ATP-binding pocket of the kinase domain. Affected individuals have overlapping phenotypes characterized by hypotonia, mild to moderate intellectual disability, behavioral disorders, and variable facial dysmorphism. Congenital heart disease occurred in six subjects; additional features present in multiple individuals included agenesis of the corpus callosum, ano-rectal malformations, seizures, and hearing or visual impairments. To evaluate the functional impact of the mutations, we measured phosphorylation at STAT1-Ser727, a known CDK8 substrate, in a CDK8 and CDK19 CRISPR double-knockout cell line transfected with wild-type (WT) or mutant CDK8 constructs. These experiments demonstrated a reduction in STAT1 phosphorylation by all mutants, in most cases to a similar extent as in a kinase-dead control. We conclude that missense mutations in CDK8 cause a developmental disorder that has phenotypic similarity to syndromes associated with mutations in other subunits of the Mediator kinase module, indicating probable overlap in pathogenic mechanisms..

Genomic alterations in cancer cells result in vulnerabilities that clinicians can exploit using molecularly targeted drugs, guided by knowledge of the tumour genotype. However, the selective activity of these drugs exerts an evolutionary pressure on cancers that can result in the outgrowth of resistant clones. Use of rational drug combinations can overcome resistance to targeted drugs, but resistance may eventually develop to combinatorial therapies. We selected MAPK- and PI3K-pathway inhibition in colorectal cancer as a model system to dissect out mechanisms of resistance. We focused on these signalling pathways because they are frequently activated in colorectal tumours, have well-characterised mutations and are clinically relevant. By treating a panel of 47 human colorectal cancer cell lines with a combination of MEK- and PI3K-inhibitors, we observe a synergistic inhibition of growth in almost all cell lines. Cells with KRAS mutations are less sensitive to PI3K inhibition, but are particularly sensitive to the combined treatment. Colorectal cancer cell lines with inherent or acquired resistance to monotherapy do not show a synergistic response to the combination treatment. Cells that acquire resistance to an MEK-PI3K inhibitor combination treatment still respond to an ERK-PI3K inhibitor regimen, but subsequently also acquire resistance to this combination treatment. Importantly, the mechanisms of resistance to MEK and PI3K inhibitors observed, MEK1/2 mutation or loss of PTEN, are similar to those detected in the clinic. ERK inhibitors may have clinical utility in overcoming resistance to MEK inhibitor regimes; however, we find a recurrent active site mutation of ERK2 that drives resistance to ERK inhibitors in mono- or combined regimens, suggesting that resistance will remain a hurdle. Importantly, we find that the addition of low concentrations of the BCL2-family inhibitor navitoclax to the MEK-PI3K inhibitor regimen improves the synergistic interaction and blocks the acquisition of resistance..

Deregulation of cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer; yet, inhibitors against these kinases are currently used only in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and the mechanisms that may undermine such dependency are poorly understood. Here, we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2 activation, involving as critical mechanistic events loss of the CDKI p21CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates the proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo. Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic..

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Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimized from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X-ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II, and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small-molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer..

BACKGROUND & AIMS: Cholangiocarcinomas (CCA) are resistant to chemotherapy, so new therapeutic agents are needed. We performed a screen to identify small-molecule compounds that are active against CCAs. Levels of microRNA 21 (MIR21 or miRNA21) are increased in CCAs. We investigated whether miRNA21 mediates resistance of CCA cells and organoids to HSP90 inhibitors. METHODS: We performed a high-throughput screen of 484 small-molecule compounds to identify those that reduced viability of 6 human CCA cell lines. We tested the effects of HSP90 inhibitors on cells with disruption of the MIR21 gene, cells incubated with MIR21 inhibitors, and stable cell lines with inducible expression of MIR21. We obtained CCA biopsies from patients, cultured them as organoids (patient-derived organoids). We assessed their architecture, mutation and gene expression patterns, response to compounds in culture, and when grown as subcutaneous xenograft tumors in mice. RESULTS: Cells with IDH1 and PBRM1 mutations had the highest level of sensitivity to histone deacetylase inhibitors. HSP90 inhibitors were effective in all cell lines, irrespective of mutations. Sensitivity of cells to HSP90 inhibitors correlated inversely with baseline level of MIR21. Disruption of MIR21 increased cell sensitivity to HSP90 inhibitors. CCA cells that expressed transgenic MIR21 were more resistant to HSP90 inhibitors than cells transfected with control vectors; inactivation of MIR21 in these cells restored sensitivity to these agents. MIR21 was shown to target the DnaJ heat shock protein family (Hsp40) member B5 (DNAJB5). Transgenic expression of DNAJB5 in CCA cells that overexpressed MIR21 re-sensitized them to HSP90 inhibitors. Sensitivity of patient-derived organoids to HSP90 inhibitors, in culture and when grown as xenograft tumors in mice, depended on expression of miRNA21. CONCLUSIONS: miRNA21 appears to mediate resistance of CCA cells to HSP90 inhibitors by reducing levels of DNAJB5. HSP90 inhibitors might be developed for the treatment of CCA and miRNA21 might be a marker of sensitivity to these agents..

Cancer cells are able to survive under conditions that cause endoplasmic reticulum stress (ER-stress), and can adapt to this stress by upregulating cell-survival signalling pathways and down-regulating apoptotic pathways. The cellular response to ER-stress is controlled by the unfolded protein response (UPR). Small Rho family GTPases are linked to many cell responses including cell growth and apoptosis. In this study, we investigate the function of small GTPases in cell survival under ER-stress. Using siRNA screening we identify that RAC1 promotes cell survival under ER-stress in cells with an oncogenic N92I RAC1 mutation. We uncover a novel connection between the UPR and N92I RAC1, whereby RAC1 attenuates phosphorylation of EIF2S1 under ER-stress and drives over-expression of ATF4 in basal conditions. Interestingly, the UPR connection does not drive resistance to ER-stress, as knockdown of ATF4 did not affect this. We further investigate cancer-associated kinase signalling pathways and show that RAC1 knockdown reduces the activity of AKT and ERK, and using a panel of clinically important kinase inhibitors, we uncover a role for MEK/ERK, but not AKT, in cell viability under ER-stress. A known major activator of ERK phosphorylation in cancer is oncogenic NRAS and we show that knockdown of NRAS in cells, which bear a Q61 NRAS mutation, sensitises to ER-stress. These findings highlight a novel mechanism for resistance to ER-stress through oncogenic activation of MEK/ERK signalling by small GTPases..

Demonstrating intracellular protein target engagement is an essential step in the development and progression of new chemical probes and potential small molecule therapeutics. However, this can be particularly challenging for poorly studied and noncatalytic proteins, as robust proximal biomarkers are rarely known. To confirm that our recently discovered chemical probe 1 (CCT251236) binds the putative transcription factor regulator pirin in living cells, we developed a heterobifunctional protein degradation probe. Focusing on linker design and physicochemical properties, we generated a highly active probe 16 (CCT367766) in only three iterations, validating our efficient strategy for degradation probe design against nonvalidated protein targets..

Patient-derived organoids (PDOs) have recently emerged as robust preclinical models; however, their potential to predict clinical outcomes in patients has remained unclear. We report on a living biobank of PDOs from metastatic, heavily pretreated colorectal and gastroesophageal cancer patients recruited in phase 1/2 clinical trials. Phenotypic and genotypic profiling of PDOs showed a high degree of similarity to the original patient tumors. Molecular profiling of tumor organoids was matched to drug-screening results, suggesting that PDOs could complement existing approaches in defining cancer vulnerabilities and improving treatment responses. We compared responses to anticancer agents ex vivo in organoids and PDO-based orthotopic mouse tumor xenograft models with the responses of the patients in clinical trials. Our data suggest that PDOs can recapitulate patient responses in the clinic and could be implemented in personalized medicine programs..

Over the past two decades there has been a great deal of interest in the development of inhibitors of the cyclin-dependent kinases (CDKs). This attention initially stemmed from observations that different CDK isoforms have key roles in cancer cell proliferation through loss of regulation of the cell cycle, a hallmark feature of cancer. CDKs have now been shown to regulate other processes, particularly various aspects of transcription. The early non-selective CDK inhibitors exhibited considerable toxicity and proved to be insufficiently active in most cancers. The lack of patient selection biomarkers and an absence of understanding of the inhibitory profile required for efficacy hampered the development of these inhibitors. However, the advent of potent isoform-selective inhibitors with accompanying biomarkers has re-ignited interest. Palbociclib, a selective CDK4/6 inhibitor, is now approved for the treatment of ER+/HER2- advanced breast cancer. Current developments in the field include the identification of potent and selective inhibitors of the transcriptional CDKs; these include tool compounds that have allowed exploration of individual CDKs as cancer targets and the determination of their potential therapeutic windows. Biomarkers that allow the selection of patients likely to respond are now being discovered. Drug resistance has emerged as a major hurdle in the clinic for most protein kinase inhibitors and resistance mechanism are beginning to be identified for CDK inhibitors. This suggests that the selective inhibitors may be best used combined with standard of care or other molecularly targeted agents now in development rather than in isolation as monotherapies..

Poor outcome for patients with glioblastomas is often associated with radioresistance. PI3K/mTOR pathway deregulation has been correlated with radioresistance; therefore, PI3K/mTOR inhibition could render tumors radiosensitive. In this study, we show that NVP-BEZ235, a dual PI3K/mTOR inhibitor, potentiates the effects of irradiation in both adult and pediatric glioblastoma cell lines, resulting in early metabolic changes detected by nuclear magnetic resonance (NMR) spectroscopy. NVP-BEZ235 radiosensitises cells to X ray exposure, inducing cell death through the inhibition of CDC25A and the activation of p21cip1(CDKN1A). Lactate and phosphocholine levels, increased with radiation, are decreased after NVP-BEZ235 and combination treatment, suggesting that inhibiting the PI3K/mTOR pathway reverses radiation induced metabolic changes. Importantly, NVP-BEZ235 potentiates the effects of irradiation in a xenograft model of adult glioblastoma, where we observed a decrease in lactate and phosphocholine levels after seven days of combination treatment. Although tumor size was not affected due to the short length of the treatment, a significant increase in CASP3 mRNA was observed in the combination group. Taken together, our data suggest that NMR metabolites could be used as biomarkers to detect an early response to combination therapy with PI3K/mTOR inhibitors and radiotherapy in adult and pediatric glioblastoma patients..

BACKGROUND: F-box and WD40 repeat domain-containing 7 (FBXW7) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of multiple oncogenic substrates. The tumour suppressor function is frequently lost in multiple cancers through genetic deletion and mutations in a broad range of tumours. Loss of FBXW7 functionality results in the stabilisation of multiple major oncoproteins, culminating in increased cellular proliferation and pro-survival pathways, cell cycle deregulation, chromosomal instability and altered metabolism. Currently, there is no therapy to specifically target FBXW7-deficient tumours. METHODS: We performed a siRNA kinome screen to identify synthetically lethal hits to FBXW7 deficiency. RESULTS: We identified and validated cyclin G-associated kinase (GAK) as a potential new therapeutic target. Combined loss of FBXW7 and GAK caused cell cycle defects, formation of multipolar mitoses and the induction of apoptosis. The synthetic lethal mechanism appears to be independent of clathrin-mediated receptor endocytosis function of GAK. CONCLUSIONS: These data suggest a putative therapeutic strategy for a large number of different types of human cancers with FBXW7 loss, many of which have a paucity of molecular abnormalities and treatment options..

Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies..

Recent experimental data showed that the PI3K pathway contributes to resistance to temozolomide (TMZ) in paediatric glioblastoma and that this effect is reversed by combination treatment of TMZ with a PI3K inhibitor. Our aim is to assess whether this combination results in metabolic changes that are detectable by nuclear magnetic resonance (NMR) spectroscopy, potentially providing metabolic biomarkers for PI3K inhibition and TMZ combination treatment. Using two genetically distinct paediatric glioblastoma cell lines, SF188 and KNS42, in vitro 1H-NMR analysis following treatment with the dual pan-Class I PI3K/mTOR inhibitor PI-103 resulted in a decrease in lactate and phosphocholine (PC) levels (P<0.02) relative to control. In contrast, treatment with TMZ caused an increase in glycerolphosphocholine (GPC) levels (P≤0.05). Combination of PI-103 with TMZ showed metabolic effects of both agents including a decrease in the levels of lactate and PC (P<0.02) while an increase in GPC (P<0.05). We also report a decrease in the protein expression levels of HK2, LDHA and CHKA providing likely mechanisms for the depletion of lactate and PC, respectively. Our results show that our in vitro NMR-detected changes in lactate and choline metabolites may have potential as non-invasive biomarkers for monitoring response to combination of PI3K/mTOR inhibitors with TMZ during clinical trials in children with glioblastoma, subject to further in vivo validation..

MYC oncoproteins deliver a potent oncogenic stimulus in several human cancers, making them major targets for drug development, but efforts to deliver clinically practical therapeutics have not yet been realized. In childhood cancer, aberrant expression of MYC and MYCN genes delineates a group of aggressive tumours responsible for a major proportion of pediatric cancer deaths. We designed a chemical-genetic screen that identifies compounds capable of enhancing proteasomal elimination of MYCN oncoprotein. We isolated several classes of compound that selectively kill MYCN expressing cells and we focus on inhibitors of PI3K/mTOR pathway in this study. We show that PI3K/mTOR inhibitors selectively killed MYCN-expressing neuroblastoma tumor cells, and induced significant apoptosis of transgenic MYCN-driven neuroblastoma tumors concomitant with elimination of MYCN protein in vivo. Mechanistically, the ability of these compounds to degrade MYCN requires complete blockade of mTOR but not PI3 kinase activity and we highlight NVP-BEZ235 as a PI3K/mTOR inhibitor with an ideal activity profile. These data establish that MYCN expression is a marker indicative of likely clinical sensitivity to mTOR inhibition, and provide a rationale for the selection of clinical candidate MYCN-destabilizers likely to be useful for the treatment of MYCN-driven cancers..

We demonstrate a designed scaffold-hop approach to the discovery of 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based dual CDK8/19 ligands. Optimized compounds in both series exhibited rapid aldehyde oxidase-mediated metabolism, which could be abrogated by introduction of an amino substituent at C5 of the 1,6-naphthyridine scaffold or at C1 of the isoquinoline scaffold. Compounds 51 and 59 were progressed to in vivo pharmacokinetic studies, and 51 also demonstrated sustained inhibition of STAT1(SER727) phosphorylation, a biomarker of CDK8 inhibition, in an SW620 colorectal carcinoma human tumor xenograft model following oral dosing..

Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors..

The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer..

It is now recognised that genetic, epigenetic and phenotypic heterogeneity within individual human cancers is responsible for therapeutic resistance - knowledge that is having a profound impact on current thinking and experimentation. There has been concern that molecularly targeted therapy is doomed to failure, with resistant clones emerging in response to the Darwinian selective pressure of any drug treatment. However, two studies have shown that the evolution of drug resistance can be restrained by co-administration of a pharmacologic inhibitor of the HSP90 molecular chaperone..

The mediator complex-associated cyclin dependent kinase CDK8 regulates β-catenin-dependent transcription following activation of WNT signaling. Multiple lines of evidence suggest CDK8 may act as an oncogene in the development of colorectal cancer. Here we describe the successful optimization of an imidazo-thiadiazole series of CDK8 inhibitors that was identified in a high-throughput screening campaign and further progressed by structure-based design. In several optimization cycles, we improved the microsomal stability, potency, and kinase selectivity. The initial imidazo-thiadiazole scaffold was replaced by a 3-methyl-1H-pyrazolo[3,4-b]-pyridine which resulted in compound 25 (MSC2530818) that displayed excellent kinase selectivity, biochemical and cellular potency, microsomal stability, and is orally bioavailable. Furthermore, we demonstrated modulation of phospho-STAT1, a pharmacodynamic biomarker of CDK8 activity, and tumor growth inhibition in an APC mutant SW620 human colorectal carcinoma xenograft model after oral administration. Compound 25 demonstrated suitable potency and selectivity to progress into preclinical in vivo efficacy and safety studies..

PURPOSE: This first-in-human dose-escalation trial evaluated the safety, tolerability, maximal-tolerated dose (MTD), dose-limiting toxicities (DLT), pharmacokinetics, pharmacodynamics, and preliminary clinical activity of pictilisib (GDC-0941), an oral, potent, and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K). PATIENTS AND METHODS: Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily, initially on days 1 to 21 every 28 days and later, using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated (18)F-FDG-PET, and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue. RESULTS: Pictilisib was well tolerated. The most common toxicities were grade 1-2 nausea, rash, and fatigue, whereas the DLT was grade 3 maculopapular rash (450 mg, 2 of 3 patients; 330 mg, 1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed >90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC >20 h·μmol/L. Significant increase in plasma insulin and glucose levels, and >25% decrease in (18)F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF-mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria, respectively. CONCLUSION: Pictilisib was safely administered with a dose-proportional pharmacokinetic profile, on-target pharmacodynamic activity at dose levels ≥100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily..

The HSP90 molecular chaperone plays a key role in the maturation, stability and activation of its clients, including many oncogenic proteins. Kinases are a substantial and important subset of clients requiring the key cochaperone CDC37. We sought an improved understanding of protein kinase chaperoning by CDC37 in cancer cells. CDC37 overexpression in human colon cancer cells increased CDK4 protein levels, which was negated upon CDC37 knockdown. Overexpressing CDC37 increased CDK4 protein half-life and enhanced binding of HSP90 to CDK4, consistent with CDC37 promoting kinase loading onto chaperone complexes. Against expectation, expression of C-terminus-truncated CDC37 (ΔC-CDC37) that lacks HSP90 binding capacity did not affect kinase client expression or activity; moreover, as with wild-type CDC37 overexpression, it augmented CDK4-HSP90 complex formation. However, although truncation blocked binding to HSP90 in cells, ΔC-CDC37 also showed diminished client protein binding and was relatively unstable. CDC37 mutants with single and double point mutations at residues M164 and L205 showed greatly reduced binding to HSP90, but retained association with client kinases. Surprisingly, these mutants phenocopied wild-type CDC37 overexpression by increasing CDK4-HSP90 association and CDK4 protein levels in cells. Furthermore, expression of the mutants was sufficient to protect kinase clients CDK4, CDK6, CRAF and ERBB2 from depletion induced by silencing endogenous CDC37, indicating that CDC37's client stabilising function cannot be inactivated by substantially reducing its direct interaction with HSP90. However, CDC37 could not compensate for loss of HSP90 function, showing that CDC37 and HSP90 have their own distinct and non-redundant roles in maintaining kinase clients. Our data substantiate the important function of CDC37 in chaperoning protein kinases. Furthermore, we demonstrate that CDC37 can stabilise kinase clients by a mechanism that is not dependent on a substantial direct interaction between CDC37 and HSP90, but nevertheless requires HSP90 activity. These results have significant implications for therapeutic targeting of CDC37..

The phosphatidylinositol-3 kinase (PI3K) pathway is one of the most frequently activated pathogenic signalling routes in human cancers, making it a rational and important target for innovative anticancer drug development and precision medicine. The three main classes of PI3K inhibitors currently in clinical testing comprise dual pan-Class I PI3K/mTOR inhibitors, pan-Class I PI3K inhibitors lacking significant mTOR activity and isoform-selective PI3K inhibitors. A major step forward in recent years is the progression of over 30 small molecule PI3K inhibitors into clinical trials and the first regulatory approval of the PI3Kδ inhibitor idelalisib for multiple B-cell malignancies. This review article focuses on the progress made in the discovery and development of novel PI3K inhibitors, with an emphasis on antitumour activity and tolerability profiles for agents that have entered clinical trials. We also discuss the key issues of drug resistance, patient selection approaches and rational targeted combinations. Finally, we envision the future development and use of PI3K inhibitors for the treatment of patients with a range of malignancies..

There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors..

WNT signaling is frequently deregulated in malignancy, particularly in colon cancer, and plays a key role in the generation and maintenance of cancer stem cells. We report the discovery and optimization of a 3,4,5-trisubstituted pyridine 9 using a high-throughput cell-based reporter assay of WNT pathway activity. We demonstrate a twisted conformation about the pyridine-piperidine bond of 9 by small-molecule X-ray crystallography. Medicinal chemistry optimization to maintain this twisted conformation, cognisant of physicochemical properties likely to maintain good cell permeability, led to 74 (CCT251545), a potent small-molecule inhibitor of WNT signaling with good oral pharmacokinetics. We demonstrate inhibition of WNT pathway activity in a solid human tumor xenograft model with evidence for tumor growth inhibition following oral dosing. This work provides a successful example of hypothesis-driven medicinal chemistry optimization from a singleton hit against a cell-based pathway assay without knowledge of the biochemical target..

The molecular chaperone heat shock protein 90 (HSP90) is required for the activity and stability of its client proteins. Pharmacologic inhibition of HSP90 leads to the ubiquitin-mediated degradation of clients, particularly activated or mutant oncogenic protein kinases. Client ubiquitination occurs via the action of one or more E3 ubiquitin ligases. We sought to identify the role of Cullin-RING family E3 ubiquitin ligases in the cellular response to HSP90 inhibition. Through a focused siRNA screen of 28 Cullin-RING ligase family members, we found that CUL5 and RBX2 were required for degradation of several HSP90 clients upon treatment of human cancer cells with the clinical HSP90 inhibitor 17-AAG. Surprisingly, silencing Cullin-5 (CUL5) also delayed the earlier loss of HSP90 client protein activity at the same time as delaying cochaperone dissociation from inhibited HSP90-client complexes. Expression of a dominant-negative CUL5 showed that NEDD8 conjugation of CUL5 is required for client degradation but not for loss of client activity or recruitment of clients and HSP90 to CUL5. Silencing CUL5 reduced cellular sensitivity to three distinct HSP90 inhibitors, across four cancer types driven by different protein kinases. Our results reveal the importance of CUL5 in multiple aspects of the cellular response to HSP90 inhibition..

The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (31P)- and proton (1H)-NMR. Similar changes were also detected using the pan-Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma..

The progressive elucidation of the molecular pathogenesis of cancer has fueled the rational development of targeted drugs for patient populations stratified by genetic characteristics. Here we discuss general challenges relating to molecular diagnostics and describe predictive biomarkers for personalized cancer medicine. We also highlight resistance mechanisms for epidermal growth factor receptor (EGFR) kinase inhibitors in lung cancer. We envisage a future requiring the use of longitudinal genome sequencing and other omics technologies alongside combinatorial treatment to overcome cellular and molecular heterogeneity and prevent resistance caused by clonal evolution..

Protein kinase clients are recruited to the Hsp90 molecular chaperone system via Cdc37, which simultaneously binds Hsp90 and kinases and regulates the Hsp90 chaperone cycle. Pharmacological inhibition of Hsp90 in vivo results in degradation of kinase clients, with a therapeutic effect in dependent tumors. We show here that Cdc37 directly antagonizes ATP binding to client kinases, suggesting a role for the Hsp90-Cdc37 complex in controlling kinase activity. Unexpectedly, we find that Cdc37 binding to protein kinases is itself antagonized by ATP-competitive kinase inhibitors, including vemurafenib and lapatinib. In cancer cells, these inhibitors deprive oncogenic kinases such as B-Raf and ErbB2 of access to the Hsp90-Cdc37 complex, leading to their degradation. Our results suggest that at least part of the efficacy of ATP-competitive inhibitors of Hsp90-dependent kinases in tumor cells may be due to targeted chaperone deprivation..

TRAIL has been shown to induce apoptosis in cancer cells, but in some cases they fail to respond to this ligand. We explored the ability of representative phosphatidylinositol-3-kinase (PI3 Kinase)/mTOR and HSP90 inhibitors to overcome TRAIL resistance by increasing apoptosis in colorectal cancer models. We determined the sensitivity of 27 human colorectal cancer and 2 non-transformed colon epithelial cell lines to TRAIL treatment. A subset of the cancer cell lines with a range of responses to TRAIL was selected from the panel for treatment with TRAIL combined with the PI3 Kinase/mTOR inhibitor PI-103 or the HSP90 inhibitor 17-AAG (tanespimycin). Two TRAIL-resistant cell lines were selected for in vivo combination studies with TRAIL and 17-AAG. We found that 13 colorectal cancer cell lines and the 2 non-transformed colon epithelial cell lines were resistant to TRAIL. We demonstrated that co-treatment of TRAIL and PI-103 or 17-AAG was synergistic or additive and significantly enhanced apoptosis in colorectal cancer cells. This was associated with decreased expression or activity of survival protein biomarkers such as ERBB2, AKT, IKKα and XIAP. In contrast, the effect of the combination treatments in non-transformed colon cells was minimal. We show here for the first time that co-treatment in vivo with TRAIL and 17-AAG in two TRAIL-resistant human colorectal cancer xenograft models resulted in significantly greater tumor growth inhibition compared to single treatments. We propose that combining TRAIL with PI3 Kinase/mTOR or HSP90 inhibitors has therapeutic potential in the treatment of TRAIL-resistant colorectal cancers..

Extraordinary progress has been made in our detailed understanding of the genetic and epigenetic mechanisms responsible for oncogenesis and cancer progression. Empowered by next-generation sequencing, many new targets and pathways have been identified to exploit oncogene and non-oncogene addiction and synthetic lethality. Kinase inhibitors feature strongly in the druggable cancer genome and 19 have been approved in oncology. While survival gains are valuable, drug resistance has emerged as the major challenge. The clonal heterogeneity and evolution of cancers is an intrinsic problem, together with feedback loops, kinase switching and activation of alternative targets and pathways. The solution to drug resistance will require the use of rationally targeted combinational regimens. The application of adaptive treatment cycles based on ongoing multi-technology profiling will be the key to long-term therapeutic success..

Inhibitors of the molecular chaperone heat shock protein 90 (HSP90) are of considerable current interest as targeted cancer therapeutic agents because of the ability to destabilize multiple oncogenic client proteins. Despite their resulting pleiotropic effects on multiple oncogenic pathways and hallmark traits of cancer, resistance to HSP90 inhibitors is possible and their ability to induce apoptosis is less than might be expected. Using an isogenic model for BAX knockout in HCT116 human colon carcinoma cells, we demonstrate the induction of BAX-dependent apoptosis at pharmacologically relevant concentrations of the HSP90 inhibitor 17-AAG both in vitro and in tumor xenografts in vivo. Removal of BAX expression by homologous recombination reduces apoptosis in vitro and in vivo but allows a lower level of cell death via a predominantly necrotic mechanism. Despite reducing apoptosis, the loss of BAX does not alter the overall sensitivity to 17-AAG in vitro or in vivo. The results indicate that 17-AAG acts predominantly to cause a cytostatic antiproliferative effect rather than cell death and further suggest that BAX status may not alter the overall clinical response to HSP90 inhibitors. Other agents may be required in combination to enhance tumor-selective killing by these promising drugs. In addition, there are implications for the use of apoptotic endpoints in the assessment of the activity of molecularly targeted agents..

The master regulator of the classical cytoprotective "heat shock" response, heat shock factor 1 (HSF1), is increasingly implicated in cancer pathogenesis, but the mechanisms remain poorly understood. A recent study connects increased protein translation to activation of HSF1 in malignant cells and demonstrates the therapeutic benefit of targeting this link..

The ribosomal P70 S6 kinases play a crucial role in PI3K/mTOR regulated signalling pathways and are therefore potential targets for the treatment of a variety of diseases including diabetes and cancer. In this study we describe the identification of three series of chemically distinct S6K1 inhibitors. In addition, we report a novel PKA-S6K1 chimeric protein with five mutations in or near its ATP-binding site, which was used to determine the binding mode of two of the three inhibitor series, and provided a robust system to aid the optimisation of the oxadiazole-substituted benzimidazole inhibitor series. We show that the resulting oxadiazole-substituted aza-benzimidazole is a potent and ligand efficient S6 kinase inhibitor, which blocks the phosphorylation of RPS6 at Ser235/236 in TSC negative HCV29 human bladder cancer cells by inhibiting S6 kinase activity and thus provides a useful tool compound to investigate the function of S6 kinases..

The molecular chaperone HSP90 maintains the activity and stability of a diverse set of "client" proteins that play key roles in normal and disease biology. Around 20 HSP90 inhibitors that deplete the oncogenic clientele have entered clinical trials for cancer. However, the full extent of the HSP90-dependent proteome, which encompasses not only clients but also proteins modulated by downstream transcriptional responses, is still incompletely characterized and poorly understood. Earlier large-scale efforts to define the HSP90 proteome have been valuable but are incomplete because of limited technical sensitivity. Here we discuss previous large-scale surveys of proteome perturbations induced by HSP90 inhibitors in light of a significant new study using state-of-the-art SILAC technology combined with more sensitive high-resolution mass spectrometry (MS) that extends the catalog of proteomic changes in inhibitor-treated cancer cells. Among wide-ranging changes, major functional responses include downregulation of protein kinase activity and the DNA damage response alongside upregulation of the protein degradation machinery. Despite this improved proteomic coverage, there was surprisingly little overlap with previous studies. This may be due in part to technical issues but is likely also due to the variability of the HSP90 proteome with the inhibitor conditions used, the cancer cell type and the genetic status of client proteins. We suggest future proteomic studies to address these factors, to help distinguish client protein components from indirect transcriptional components and to address other key questions in fundamental and translational HSP90 research. Such studies should also reveal new biomarkers for patient selection and novel targets for therapeutic intervention..

The discovery and development of small molecule cancer drugs has been revolutionised over the last decade. Most notably, we have moved from a one-size-fits-all approach that emphasized cytotoxic chemotherapy to a personalised medicine strategy that focuses on the discovery and development of molecularly targeted drugs that exploit the particular genetic addictions, dependencies and vulnerabilities of cancer cells. These exploitable characteristics are increasingly being revealed by our expanding understanding of the abnormal biology and genetics of cancer cells, accelerated by cancer genome sequencing and other high-throughput genome-wide campaigns, including functional screens using RNA interference. In this review we provide an overview of contemporary approaches to the discovery of small molecule cancer drugs, highlighting successes, current challenges and future opportunities. We focus in particular on four key steps: Target validation and selection; chemical hit and lead generation; lead optimization to identify a clinical drug candidate; and finally hypothesis-driven, biomarker-led clinical trials. Although all of these steps are critical, we view target validation and selection and the conduct of biology-directed clinical trials as especially important areas upon which to focus to speed progress from gene to drug and to reduce the unacceptably high attrition rate during clinical development. Other challenges include expanding the envelope of druggability for less tractable targets, understanding and overcoming drug resistance, and designing intelligent and effective drug combinations. We discuss not only scientific and technical challenges, but also the assessment and mitigation of risks as well as organizational, cultural and funding problems for cancer drug discovery and development, together with solutions to overcome the 'Valley of Death' between basic research and approved medicines. We envisage a future in which addressing these challenges will enhance our rapid progress towards truly personalised medicine for cancer patients..

The objective of the ONCODEATH consortium [EU Research Consortium "ONCODEATH" (2006-2010)] was to achieve sensitization of solid tumor cells to death receptor related therapies using rational mechanism-based drug combinations of targeted therapies. In this collaborative effort, during a period of 42 mo, cell and animal model systems of defined oncogenes were generated. Exploitation of generated knowledge and tools enabled the consortium to achieve the following research objectives: (1) elucidation of tumor components which confer sensitivity or resistance to TRAIL-induced cell death; (2) providing detailed knowledge on how small molecule Hsp90, Aurora, Choline kinase, BRAF inhibitors, DNA damaging agents, HDAC and DNMT inhibitors affect the intrinsic apoptotic amplification and execution machineries; (3) optimization of combined action of TRAIL with these therapeutics for optimum effects with minimum concentrations and toxicity in vivo. These findings provide mechanistic basis for a pharmacogenomic approach, which could be exploited further therapeutically, in order to reach novel personalized therapies for cancer patients..

Evidence that the phosphoinositide 3-kinase (PI3K) pathway is deregulated in ovarian cancer is largely based on the analysis of surgical specimens sampled at diagnosis and may not reflect the biology of advanced ovarian cancer. We aimed to investigate PI3K signaling in cancer cells isolated from patients with advanced ovarian cancer. Ascites samples were analyzed from 88 patients, of whom 61 received further treatment. Cancer cells were immunomagnetically separated from ascites, and the signaling output of the PI3K pathway was studied by quantifying p-AKT, p-p70S6K, and p-GSK3β by ELISA. Relevant oncogenes, such as PIK3CA and AKT, were sequenced by PCR-amplified mass spectroscopy detection methods. In addition, PIK3CA and AKT2 amplifications and PTEN deletions were analyzed by FISH. p-p70S6K levels were significantly higher in cells from 37 of 61 patients who did not respond to subsequent chemotherapy (0.7184 vs. 0.3496; P = 0.0100), and this difference was greater in patients who had not received previous chemotherapy. PIK3CA and AKT mutations were present in 5% and 0% of samples, respectively. Amplification of PIK3CA and AKT2 and deletion of PTEN was seen in 10%, 10%, and 27% of samples, respectively. Mutations of PIK3CA and amplification of PIK3CA/AKT2 or deletion of PTEN did not correlate with levels of p-AKT, p-p70S6K, and p-GSK3β. In patients with advanced ovarian cancer, there is an association between levels of p-p70S6K and response to subsequent chemotherapy. There is no clear evidence that this is driven specifically by PIK3CA or AKT mutations or by amplifications or deletion of PTEN..

Two recent papers published in Nature demonstrate the power of systematic high-throughput pharmacologic profiling of very large, diverse, molecularly-characterized human cancer cell line panels to reveal linkages between genetic profile and targeted-drug sensitivity. Known oncogene addictions are confirmed while surprising complexities and biomarker relationships with clinical potential are revealed..

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The discovery and clinical development of small-molecule inhibitors of the phosphatidylinositide 3-kinase (PI3 kinase) family of lipid kinases have marked a remarkable 20-year journey that follows the progressive developments in cancer biology over the last few decades: from hypothesis-driven, basic cancer research that began with viral oncogenesis and developed in the 1960s and 70s, through the discovery of individual mutated oncogenes and tumor suppressor genes in 1970 and 80s and the linkage of these cancer genes to signal transduction pathways in the 1990s, to all large-scale genome-wide sequencing, functional screening, and network biology efforts today. Thus, PI3 kinase research began with the discovery in 1985 of a new type of enzyme activity associated with viral oncogenesis. It benefited greatly from the discovery of wortmannin and LY294002 as PI3 kinase inhibitors and chemical tools in late 1980s to mid-90s. Alongside these tools, genetic validation of PI3 kinase as a target initially involved activation by upstream oncogenic receptor tyrosine kinases and RAS mutation, together with overexpression and amplification of the p110α catalytic isoform of PI3 kinase and frequent loss of the tumor suppressor and negative regulator of PI3 kinase activity, PTEN. As PI3 kinase drug development began, further stimulus came from the discovery through genome sequencing of mutations in PIK3CA, which encodes p110α and is the most frequently mutated kinase in the human genome. From these beginnings, there are now many PI3 kinase inhibitors in clinical trials and more in preclinical development. We review progress, current challenges, and future opportunities in this article..

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Despite the promise of the new generation of molecularly targeted drugs, intrinsic and acquired resistance is proving to be as problematic as with cytotoxic drugs. Two recent papers have identified novel ways by which non-small cell lung cancers can exhibit resistance to EGFR inhibitors and suggest new therapeutic workarounds..

Two classes of trisubstituted pyrimidines related to PI-103 1 have been prepared and their inhibitory activities against phosphatidylinositol 3-kinase (PI3K) p110α were determined. From those with direct 6-aryl substitution compound 11a was the most potent inhibitor with an IC₅₀ value of 62 nM, and showed similar activity against other class 1a PI3K isoforms tested, p110β and p110γ. When a linking chain was introduced, as in the second exemplified class, compound 15f inhibited p110α with IC₅₀ 142 nM, and showed greater selectivity towards p110α. Compounds of both classes showed promising inhibition of cellular proliferation in IGROV-1 ovarian cancer cells. Among compounds designed to replace the 3-phenolic motif with structural isosteres, analogues incorporating a 4-indazolyl group possessed enzyme and cellular activities comparable to the parent phenols..

The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases. Since the mid-late 1990s, considerable progress has been made in the discovery and development of small molecule ATP-competitive PI3K inhibitors, a number of which have entered early phase human trials over recent years from which key clinical results are now being disclosed. This review summarizes progress made to date, primarily on the discovery and characterization of class I and dual class I/IV subtype inhibitors, together with advances that have been made in translational and clinical research, notably in cancer..

The phosphoinositide 3-kinases (PI3Ks) constitute an important family of lipid kinase enzymes that control a range of cellular processes through their regulation of a network of signal transduction pathways, and have emerged as important therapeutic targets in the context of cancer, inflammation and cardiovascular diseases. Since the mid-late 1990s, considerable progress has been made in the discovery and development of small molecule ATP-competitive PI3K inhibitors, a number of which have entered early phase human trials over recent years from which key clinical results are now being disclosed. This review summarizes progress made to date, primarily on the discovery and characterization of class I and dual class I/IV subtype inhibitors, together with advances that have been made in translational and clinical research, notably in cancer..

The phosphoinositide 3-kinase (PI3K) pathway is a major target for cancer drug development. PI-103 is an isoform-selective class I PI3K and mammalian target of rapamycin inhibitor. The aims of this work were as follows: first, to use magnetic resonance spectroscopy (MRS) to identify and develop a robust pharmacodynamic (PD) biomarker for target inhibition and potentially tumor response following PI3K inhibition; second, to evaluate mechanisms underlying the MRS-detected changes. Treatment of human PTEN null PC3 prostate and PIK3CA mutant HCT116 colon carcinoma cells with PI-103 resulted in a concentration- and time-dependent decrease in phosphocholine (PC) and total choline (tCho) levels (P < 0.05) detected by phosphorus ((31)P)- and proton ((1)H)-MRS. In contrast, the cytotoxic microtubule inhibitor docetaxel increased glycerophosphocholine and tCho levels in PC3 cells. PI-103-induced MRS changes were associated with alterations in the protein expression levels of regulatory enzymes involved in lipid metabolism, including choline kinase alpha (ChoK(alpha)), fatty acid synthase (FAS), and phosphorylated ATP-citrate lyase (pACL). However, a strong correlation (r(2) = 0.9, P = 0.009) was found only between PC concentrations and ChoK(alpha) expression but not with FAS or pACL. This study identified inhibition of ChoK(alpha) as a major cause of the observed change in PC levels following PI-103 treatment. We also showed the capacity of (1)H-MRS, a clinically well-established technique with higher sensitivity and wider applicability compared with (31)P-MRS, to assess response to PI-103. Our results show that monitoring the effects of PI3K inhibitors by MRS may provide a noninvasive PD biomarker for PI3K inhibition and potentially of tumor response during early-stage clinical trials with PI3K inhibitors..

The phosphatidylinositide 3-kinase (PI3K) pathway is very commonly activated in a wide range of human cancers and is a major driving force in oncogenesis. One of the class I lipid kinase members of the PI3K family, p110alpha, is probably the most commonly mutated kinase in the human genome. Alongside genetic, molecular biological, and biochemical studies, chemical inhibitors have been extremely helpful tools in understanding the role of PI3K enzymes in signal transduction and downstream physiological and pathological processes, and also in validating PI3Ks as therapeutic targets. Although they have been valuable in the past, the early and still frequently employed inhibitors, wortmannin and LY294002, have significant limitations as chemical tools. Here, we discuss the case history of the discovery and properties of an increasingly used chemical probe, the pan-class I PI3K and mammalian target of rapamycin (mTOR) inhibitor PI-103 (a pyridofuropyrimidine), and its very recent evolution into the thienopyrimidine drug GDC-0941, which exhibits excellent oral anticancer activity in preclinical models and is now undergoing phase I clinical trials in cancer patients. We also illustrate the impact of structural biology on the design of PI3K inhibitors and on the interpretation of their effects. The challenges and outlook for drugging the PI3 kinome are discussed in the more general context of the role of structural biology and chemical biology in innovative drug discovery..

Gliomas are primary brain tumors with poor prognosis that exhibit frequent abnormalities in phosphatidylinositol 3-kinase (PI3 kinase) signaling. We investigated the molecular mechanism of action of the isoform-selective class I PI3 kinase and mTOR inhibitor PI-103 in human glioma cells. The potent inhibitory effects of PI-103 on the PI3 kinase pathway were quantified. PI-103 and the mTOR inhibitor rapamycin both inhibited ribosomal protein S6 phosphorylation but there were clear differences in the response of upstream components of the PI3 kinase pathway, such as phosphorylation of Thr(308)-AKT, that were inhibited by PI-103 but not rapamycin. Gene expression profiling identified altered expression of genes encoding regulators of the cell cycle and cholesterol metabolism, and genes modulated by insulin or IGF1 signaling, rapamycin treatment or nutrient starvation. PI-103 decreased expression of positive regulators of G(1)/S phase progression and increased expression of the negative cell cycle regulator p27(kip1). A reversible PI-103-mediated G(1) cell cycle arrest occurred without significant apoptosis, consistent with the altered gene expression detected. PI-103 induced vacuolation and processing of LC-3i to LC-3ii, which are features of an autophagic response. In contrast to PI-103, LY294002 and PI-387 induced apoptosis, indicative of likely off-target effects. PI-103 interacted synergistically or additively with cytotoxic agents used in the treatment of glioma, namely vincristine, BCNU and temozolomide. Compared to individual treatments, the combination of PI-103 with temozolomide significantly improved the response of U87MG human glioma xenografts. Our results support the therapeutic potential for PI3 kinase inhibitors with a PI-103-like profile as therapeutic agents for the treatment of glioma..

A dose-escalation, phase I study evaluated the safety, pharmacokinetics, pharmacogenomics, and efficacy of ES-285, a novel agent isolated from a marine mollusc, in adult cancer patients. Patients received a 24-hour i.v. infusion of ES-285 once every 3 weeks until disease progression or unacceptable toxicity. The starting dose was 4 mg/m(2). Dose escalation in cohorts of at least three patients proceeded according to the worst toxicity observed in the previous cohort. Twenty-eight patients were treated with 72 courses of ES-285 across eight dose levels. No dose-limiting toxicities were seen between 4 and 128 mg/m(2). Two of four patients treated at 256 mg/m(2) had dose-limiting reversible grade 3 transaminitis; one patient at 256 mg/m(2) also had transient grade 3 central neurotoxicity. One of three patients subsequently treated at 200 mg/m(2) died following drug-related central neurotoxicity. Other toxicities included phlebitis, nausea, fatigue, and fever. Pharmacokinetic studies indicated dose proportionality with high volume of distribution (median V(ss) at 256 mg/m(2) was 2,389 liters; range, 1,615-4,051 liters) and long elimination half life (median t(1/2) at 256 mg/m(2) was 28 h; range, 21-32 h). The three patients with dose-limiting toxicity had the highest drug exposure. Pharmacogenomic studies of paired surrogate tissue samples identified changes in gene expression following treatment that correlated with increasing dose. Disease stabilization for 6 to 18 weeks was recorded in nine patients. Using this schedule, 128 mg/m(2) was considered safe and feasible. At this dose, pharmacologically relevant concentrations of the drug were safely achieved with pharmacogenomic studies indicating changes in the expression of genes of potential mechanistic relevance..

The phosphatidylinositide 3-kinase pathway is frequently deregulated in human cancers and inhibitors offer considerable therapeutic potential. We previously described the promising tricyclic pyridofuropyrimidine lead and chemical tool compound PI-103. We now report the properties of the pharmaceutically optimized bicyclic thienopyrimidine derivatives PI-540 and PI-620 and the resulting clinical development candidate GDC-0941. All four compounds inhibited phosphatidylinositide 3-kinase p110alpha with IC(50) < or = 10 nmol/L. Despite some differences in isoform selectivity, these agents exhibited similar in vitro antiproliferative properties to PI-103 in a panel of human cancer cell lines, with submicromolar potency in PTEN-negative U87MG human glioblastoma cells and comparable phosphatidylinositide 3-kinase pathway modulation. PI-540 and PI-620 exhibited improvements in solubility and metabolism with high tissue distribution in mice. Both compounds gave improved antitumor efficacy over PI-103, following i.p. dosing in U87MG glioblastoma tumor xenografts in athymic mice, with treated/control values of 34% (66% inhibition) and 27% (73% inhibition) for PI-540 (50 mg/kg b.i.d.) and PI-620 (25 mg/kg b.i.d.), respectively. GDC-0941 showed comparable in vitro antitumor activity to PI-103, PI-540, and PI-620 and exhibited 78% oral bioavailability in mice, with tumor exposure above 50% antiproliferative concentrations for >8 hours following 150 mg/kg p.o. and sustained phosphatidylinositide 3-kinase pathway inhibition. These properties led to excellent dose-dependent oral antitumor activity, with daily p.o. dosing at 150 mg/kg achieving 98% and 80% growth inhibition of U87MG glioblastoma and IGROV-1 ovarian cancer xenografts, respectively. Together, these data support the development of GDC-0941 as a potent, orally bioavailable inhibitor of phosphatidylinositide 3-kinase. GDC-0941 has recently entered phase I clinical trials..

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The cochaperone CDC37 promotes the association of HSP90 with the protein kinase subset of client proteins to maintain their stability and signalling functions. HSP90 inhibitors induce depletion of clients, which include several oncogenic kinases. We hypothesized that the targeting of CDC37 using siRNAs would compromise the maturation of these clients and increase the sensitivity of cancer cells to HSP90 inhibitors. Here, we show that silencing of CDC37 in human colon cancer cells diminished the association of kinase clients with HSP90 and reduced levels of the clients ERBB2, CRAF, CDK4 and CDK6, as well as phosphorylated AKT. CDC37 silencing promoted the proteasome-mediated degradation of kinase clients, suggesting a degradation pathway independent from HSP90 binding. Decreased cell signalling through kinase clients was also demonstrated by reduced phosphorylation of downstream substrates and colon cancer cell proliferation was subsequently reduced by the inhibition of the G1/S-phase transition. Furthermore, combining CDC37 silencing with the HSP90 inhibitor 17-AAG induced more extensive and sustained depletion of kinase clients and potentiated cell cycle arrest and apoptosis. These results support an essential role for CDC37 in concert with HSP90 in maintaining oncogenic protein kinase clients and endorse the therapeutic potential of targeting CDC37 in cancer..

Heat-shock protein 70 (HSP70) isoforms contribute to tumorigenesis through their well-documented antiapoptotic activity and via their role as cochaperones for the HSP90 molecular chaperone. HSP70 expression is induced following treatment with HSP90 inhibitors, which may attenuate the cell death effects of this class of inhibitor. Here we show that silencing either heat-shock cognate 70 (HSC70) or HSP72 expression in human cancer cell lines has no effect on HSP90 activity or cell proliferation. However, simultaneously reducing the expression of both of these isoforms induces proteasome-dependent degradation of HSP90 client proteins, G1 cell-cycle arrest, and extensive tumor-specific apoptosis. Importantly, simultaneous silencing of HSP70 isoforms in nontumorigenic cell lines does not result in comparable growth arrest or induction of apoptosis, indicating a potential therapeutic window..

Activation of protein kinase clients by the Hsp90 system is mediated by the cochaperone protein Cdc37. Cdc37 requires phosphorylation at Ser13, but little is known about the regulation of this essential posttranslational modification. We show that Ser13 of uncomplexed Cdc37 is phosphorylated in vivo, as well as in binary complex with a kinase (C-K), or in ternary complex with Hsp90 and kinase (H-C-K). Whereas pSer13-Cdc37 in the H-C-K complex is resistant to nonspecific phosphatases, it is efficiently dephosphorylated by the chaperone-targeted protein phosphatase 5 (PP5/Ppt1), which does not affect isolated Cdc37. We show that Cdc37 and PP5/Ppt1 associate in Hsp90 complexes in yeast and in human tumor cells, and that PP5/Ppt1 regulates phosphorylation of Ser13-Cdc37 in vivo, directly affecting activation of protein kinase clients by Hsp90-Cdc37. These data reveal a cyclic regulatory mechanism for Cdc37, in which its constitutive phosphorylation is reversed by targeted dephosphorylation in Hsp90 complexes..

The promising antitumor activity of 17-allylamino-17-demethoxygeldanamycin (17AAG) results from inhibition of the molecular chaperone heat shock protein 90 (HSP90) and subsequent degradation of multiple oncogenic client proteins. Gene expression microarray and proteomic analysis were used to profile molecular changes in the A2780 human ovarian cancer cell line treated with 17AAG. Comparison of results with an inactive analogue and an alternative HSP90 inhibitor radicicol indicated that increased expression of HSP72, HSC70, HSP27, HSP47, and HSP90beta at the mRNA level were on-target effects of 17AAG. HSP27 protein levels were increased in tumor biopsies following treatment of patients with 17AAG. A group of MYC-regulated mRNAs was decreased by 17AAG. Of particular interest and novelty were changes in expression of chromatin-associated proteins. Expression of the heterochromatin protein 1 was increased, and expression of the histone acetyltransferase 1 and the histone arginine methyltransferase PRMT5 was decreased by 17AAG. PRMT5 was shown to be a novel HSP90-binding partner and potential client protein. Cellular protein acetylation was reduced by 17AAG, which was shown to have an antagonistic interaction on cell proliferation with the histone deacetylase inhibitor trichostatin A. This mRNA and protein expression analysis has provided new insights into the complex molecular pharmacology of 17AAG and suggested new genes and proteins that may be involved in response to the drug or be potential biomarkers of drug action..

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development..

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Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC50 values against recombinant PI3K isoforms p110alpha (2 nmol/L), p110beta (3 nmol/L), p110delta (3 nmol/L), and p110gamma (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 micromol/L and exhibited an IC50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic..

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High-throughput screening of chemical libraries and the subsequent rapid progress of hit compounds through an iterative developmental test cascade are essential parts of modern molecular mechanism-based drug discovery. These processes depend on the use of efficient assay technologies and equipment. Enzyme-linked immunosorbent assays have historically been carried out in 96-well microtitre plates. Improvements in reagents and assay technologies mean that solid-phase immunoassays can be adapted for higher throughput to play an important role in modern drug discovery. The molecular chaperone heat-shock protein (Hsp) 90 is an important anticancer drug target because it maintains the conformation, stability, and function of many important oncogenic client proteins, including those involved with signal transduction, cell proliferation, survival, differentiation, motility angiogenesis, and metastasis. Using the standard inhibitors of the adenosine triphosphatase (ATPase) activity of Hsp90, geldanamycin (GA) and 17-allylamino-17- demethoxygeldanamycin (17AAG), novel solid-phase immunoassays have been validated using a time-resolved fluorescence (TRF) end point. Their utility for confirming the mechanism of action of Hsp90 inhibition in secondary cell-based assays has been shown and applied to the novel Hsp90 inhibitor CCT018159. Adaptation of these assays for later studies using human tumour xenografts and samples obtained from a Phase 1 trial of 17AAG is also described. Finally, comparison is made between the use and applicability of this type of immunoassay and other techniques such as western blotting, immunohistochemistry, and flow cytometry analysis..

We review in detail how gene expression microarray technology is benefiting all phases of the discovery, development and subsequent use of new cancer therapeutics. Global gene expression profiling is valuable in cancer classification, elucidation of biochemical pathways and the identification of potential targets for novel molecular therapeutics. We exemplify the value in tissue culture and animal models of cancer, as well as in clinical studies. The power of expression profiling alongside gene knockout or knockdown methods such as RNA interference is illustrated. The use of basal or constitutive gene expression profiling to understand and predict drug sensitivity or resistance is described. The ability of expression profiling to define detailed molecular signatures of drug action is emphasised. The approach can identify on-target and off-target effects. It can be used to identify molecular biomarkers for proof of concept studies, pharmacodynamic endpoints and prognostic markers for predicting outcome and patient selection..

The detailed molecular basis and determinants of in vivo tumour sensitivity to conventional anticancer agents remain unclear. We examined the cellular and molecular consequences of cisplatin treatment using two ovarian tumour xenograft models that had not been previously adapted to culture in vitro. Both xenografts were curable with clinically relevant multiple doses of cisplatin. Following a single dose of cisplatin (6 mg kg(-1) i.p.) growth delays of 25 and 75 days were obtained for pxn100 and pxn65, respectively. This difference in response was not due to differences in DNA damage. Pxn100 tumours had a functional p53 response and a wild-type p53 sequence, whereas pxn65 harboured a mutant p53 and lacked a functional p53 response. Microarray analysis revealed the induction of p53-regulated genes and regulators of checkpoint control and apoptosis in pxn100 tumours following cisplatin-treatment. By contrast, there was no p53-dependent response and only limited changes in gene expression were detected in the pxn65 tumours. TUNEL analysis demonstrated high levels of apoptosis in the pxn100 tumours following cisplatin treatment, but there was no detectable apoptosis in the pxn65 tumours. Our observations show that a marked in vivo response to cisplatin can occur via p53-dependent apoptosis or independently of p53 status in human ovarian xenografts..

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HSP90 inhibitors such as 17AAG have the major therapeutic advantage that they exert downstream inhibitory effects on multiple oncogenic client proteins. They therefore block several mission critical cancer-causing pathways and have the potential to modulate all of the hallmark biological features of malignancy. Consistent with this combinatorial anti-oncogenic profile, 17AAG exhibits broad-spectrum antitumour activity against cultured cancer cell lines and in vivo animal models. However, there are clear differences in sensitivity between various cancer cell lines and it is quite possible that some tumour types or individual patients will be more responsive in the clinic than others. We describe the methods used to investigate the genes and proteins involved in the mechanism of action of HSP90 inhibitors and discuss the significance of these for cellular sensitivity. Methods used involve the conventional cell and molecular biology techniques, together with the more recent application of high throughput global technologies such as gene expression microarrays and proteomics. Selected examples that seem to play a role in sensitivity to HSP90 inhibitors are highlighted and the potential relevance to the response of cancer patients is discussed. Important determinants of response include: 1) Dependence upon key HSP90 client proteins such as ERBB2, steroid hormone receptors and AKT/PKB; 2) Levels of HSP90 family members and co-chaperones, such as HSP70 and AHA1; and 3) expression of various cell cycle and apoptotic regulators. In the case of 17AAG, metabolic enzymes such as NQO1 and membrane efflux pumps are also important for sensitivity..

Global gene expression profiling has potential for elucidating the complex cellular effects and mechanisms of action of novel targeted anticancer agents or existing chemotherapeutics for which the precise molecular mechanism of action may be unclear. In this study, decreased expression of genes required for RNA and protein synthesis, and for metabolism were detected in rectal cancer biopsies taken from patients during a 5-fluorouracil infusion. Our observations demonstrate that this approach is feasible and can detect responses that may have otherwise been missed by conventional methods. The results suggested new mechanism-based combination treatments for colorectal cancer and demonstrated that expression profiling could provide valuable information on the molecular pharmacology of established and novel drugs..

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Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle..

Magnetic resonance spectroscopy is increasingly used as a non-invasive method to investigate apoptosis. Apoptosis was induced in Jurkat T-cells by Fas mAb. (1)H magnetic resonance spectra of live cells showed an increase in methylene signal as well as methylene/methyl ratio of fatty acid side chains at 5 and 24 h following induction of apoptosis. To explain this observation, (1)H magnetic resonance spectra of cell extracts were investigated. These demonstrated a 70.0+/-7.0%, 114.0+/-8.0% and 90.0+/-5.0% increase in the concentration of triacylglycerols following 3, 5 and 7 h of Fas mAb treatment (P<0.05). Confocal microscopy images of cells stained with the lipophilic dye Nile Red demonstrated the presence of lipid droplets in the cell cytoplasm. Quantification of the stained lipids by flow cytometry showed a good correlation with the magnetic resonance results (P > or =0.05 at 3, 5 and 7 h). (31)P magnetic resonance spectra showed a drop in phosphatidylcholine content of apoptosing cells, indicating that alteration in phosphatidylcholine metabolism could be the source of triacylglycerol accumulation during apoptosis. In summary, apoptosis is associated with an early accumulation of mobile triacylglycerols mostly in the form of cytoplasmic lipid droplets. This is reflected in an increase in the methylene/methyl ratio which could be detected by magnetic resonance spectroscopy..

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BACKGROUND: Kirsten ras (Ki-ras) mutations are common in gastrointestinal cancer and one codon 12 mutation, glycine to valine, is particularly aggressive in colorectal cancer. AIMS: To investigate if this valine point mutation could be targeted with antisense oligonucleotides and to determine the efficacy of any antisense/mRNA interaction. METHODS: Twenty nine antisense oligonucleotides were screened against target and control Ki-ras RNA in a cell free system and against target and control cell lines in culture. RESULTS: The activity and specificity of the oligonucleotides varied. Results for the individual oligonucleotides were consistent in a cell free model and in cell culture using two different uptake promoters. Only one oligonucleotide was specific in its cleavage of target Ki-ras mRNA in the cell free system and appeared specific in cell culture, although changes in Ki-ras mRNA and protein expression following a single treatment could not be detected. Experiments in the cell free system showed that the point mutation is relatively inaccessible to oligonucleotides. Other sites on the Ki-ras RNA molecule, away from the point mutation, can be targeted more effectively. CONCLUSIONS: Successful targeting of the clinically relevant Ki-ras point mutation with antisense oligonucleotides is difficult because of RNA structure at the mutated site and is inefficient compared with other sites on the Ki-ras mRNA..

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Kirsten-ras is frequently mutated in colorectal cancers and may be an important therapeutic target, particularly because we have previously shown that acquisition of a mutation is associated with a poorer outcome. Understanding the role of Kirsten-ras and the consequences of inhibiting its activity or expression will contribute to our comprehension of colorectal cancer biology and may help to rationalize the choice of molecular targets suitable for therapeutic manipulation. Therefore we undertook a simple screen, incubating a library of oligonucleotides with Kirsten-ras mRNA and RNase H to identify an antisense oligonucleotide that effectively inhibited Kirsten-ras expression. We show for the first time in a human colon cancer cell line that inhibition of Kirsten-ras expression inhibits constitutive phosphorylation of Erk1/2, but not c-Akt, suggesting that in these cells constitutive phosphorylation of Erk 1/2 is dependent upon Kirsten-ras. Successful inhibition of Kirsten-ras had little effect on cell number or cell death and there was no evidence for accumulation of cells in any particular phase of the cell cycle. Kirsten-ras inhibition significantly reduced secretion of VEGF-A165 into the culture medium. Gene expression profiling by microarray detected altered expression of a number of genes. Of particular interest for future studies was the altered expression of genes encoding products involved in protein trafficking and the potential effects of these changes on cell adhesion. Our results suggest that, at least in this model, Kirsten-ras may contribute to malignancy predominantly through effects on angiogenesis, invasion, and metastasis, and that therapies directed at Kirsten-ras, including antisense approaches, may have particular utility through these mechanisms..

Antitumor and pharmacodynamic studies were performed in MCF-7 human breast cancer cells and companion xenografts with the farnesyl protein transferase inhibitor, R115777, presently undergoing Phase II clinical trials, including in breast cancer. R115777 inhibited growth of MCF-7 cells in vitro with an IC(50) of 0.31 +/- 0.25 microM. Exposure of MCF-7 cells to increasing concentrations of R115777 for 24 h resulted in the inhibition of protein farnesylation, as indicated by the appearance of prelamin A at concentrations >1 microM. After continuous exposure to 2 microM R115777, prelamin A levels peaked at 2 h post drug exposure and remained high for up to 72 h. R115777 administered p.o. twice daily for 10 consecutive days to mice bearing established s.c. MCF-7 xenografts induced tumor inhibition at a dose of 25 mg/kg [percentage of treated versus control (% T/C) = 63% at day 21]. Greater inhibition was observed at doses of 50 mg/kg (% T/C at day 21 = 38%) or 100 mg/kg (% T/C at day 21 = 43%). The antitumor effect appeared to be mainly cytostatic with little evidence of tumor shrinkage to less than the starting volume. Tumor response correlated with an increase in the appearance of prelamin A, but no changes in the prenylation of lamin B, heat shock protein 40, or N-Ras were detectable. In addition, significant increases in apoptotic index and p21(WAF1/CIP1) expression were observed, concomitant with a decrease in proliferation as measured by Ki-67 staining. An increase in prelamin A was also observed in peripheral blood lymphocytes in a breast cancer patient who responded to R115777. These data show that R115777 possesses preclinical antitumor activity against human breast cancer and that the appearance of prelamin A may provide a sensitive and convenient pharmacodynamic marker of inhibition of prenylation and/or response..

With the imminent completion of the Human Genome Project, biomedical research is being revolutionised by the ability to carry out investigations on a genome wide scale. This is particularly important in cancer, a disease that is caused by accumulating abnormalities in the sequence and expression of a number of critical genes. Gene expression microarray technology is gaining increasingly widespread use as a means to determine the expression of potentially all human genes at the level of messenger RNA. In this commentary, we review developments in gene expression microarray technology and illustrate the progress and potential of the methodology in cancer biology, pharmacology, and drug development. Important applications include: (a) development of a more global understanding of the gene expression abnormalities that contribute to malignant progression; (b) discovery of new diagnostic and prognostic indicators and biomarkers of therapeutic response; (c) identification and validation of new molecular targets for drug development; (d) provision of an improved understanding of the molecular mode of action during lead identification and optimisation, including structure-activity relationships for on-target versus off-target effects; (e) prediction of potential side-effects during preclinical development and toxicology studies; (f) confirmation of a molecular mode of action during hypothesis-testing clinical trials; (g) identification of genes involved in conferring drug sensitivity and resistance; and (h) prediction of patients most likely to benefit from the drug and use in general pharmacogenomic studies. As a result of further technological improvements and decreasing costs, the use of microarrays will become an essential and potentially routine tool for cancer and biomedical research..

17-Allylamino-17-demethoxygeldanamycin (17AAG) is a first-in-class heat shock protein 90 (Hsp90) molecular chaperone inhibitor to enter clinical trials. The downstream molecular and cellular consequences of Hsp90 inhibition are not well defined. 17AAG has shown activity against human colon cancer in cell culture and xenograft models. In this study, we demonstrated that in addition to depleting c-Raf-1 and inhibiting ERK-1/2 phosphorylation in human colon adenocarcinoma cells, 17AAG also depleted N-ras, Ki-ras, and c-Akt and inhibited phosphorylation of c-AKT: A consequence of these events was the induction of cell line-dependent cytostasis and apoptosis, although the latter did not result from dephosphorylation of proapoptotic BAD: One cell line, KM12, did not exhibit apoptosis and in contrast to the other cell lines overexpressed Bag-1, but did not express BAX: Taken together with other determinants of 17AAG sensitivity, these results should contribute to a more complete understanding of the molecular pharmacology of 17AAG, which in turn should aid the future rational clinical development and use of the drug in colon and other tumor types..

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PURPOSE: To evaluate the pharmacokinetics and toxicity of an antisense oligonucleotide targeting bcl-2 in patients with non-Hodgkin's lymphoma (NHL) and to determine efficacy using clinical and biologic end points. PATIENTS AND METHODS: Twenty-one patients with Bcl-2-positive relapsed NHL received a 14-day subcutaneous infusion of G3139, an 18-mer phosphorothioate oligonucleotide complementary to the first six codons of the bcl-2 open reading frame. Plasma pharmacokinetics were measured by anion exchange high-performance liquid chromatography. Response was assessed by computed tomography. Changes in Bcl-2 expression were measured by fluorescence-activated cell sorting of patients' tumor samples. RESULTS: Eight cohorts of patients received doses between 4. 6 and 195.8 mg/m(2)/d. No significant systemic toxicity was seen at doses up to 110.4 mg/m(2)/d. All patients displayed skin inflammation at the subcutaneous infusion site. Dose-limiting toxicities were thrombocytopenia, hypotension, fever, and asthenia. The maximum-tolerated dose was 147.2 mg/m(2)/d. Plasma levels of G3139 equivalent to the efficacious plasma concentration in in vivo models were produced with doses above 36.8 mg/m(2)/d. Plasma levels associated with dose-limiting toxicity were greater than 4 microg/mL. By standard criteria, there was one complete response, 2 minor responses, nine cases of stable disease, and nine cases of progressive disease. Bcl-2 protein was reduced in seven of 16 assessable patients. This reduction occurred in tumor cells derived from lymph nodes in two patients and from peripheral blood or bone marrow mononuclear cell populations in the remaining five patients. CONCLUSION: Bcl-2 antisense therapy is feasible and shows potential for antitumor activity in NHL. Downregulation of Bcl-2 protein suggests a specific antisense mechanism..

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A number of molecular therapeutic agents, derived from exploiting our knowledge of the oncogenic pathways that are frequently deregulated in cancer, are now entering clinical trials. One of these is the novel agent 17-allylamino-17-demethoxygeldanamycin that acts to inhibit the hsp90 molecular chaperone. Treatment of four human colon cancer cell lines with iso-effective concentrations of this agent resulted in depletion of c-raf-1 and akt and inhibition of signal transduction. We have used gene expression array analysis to identify genes responsive to treatment with this drug. The expression of hsp90 client protein genes was not affected, but hsc hsp70, hsp90beta, keratin 8, keratin 18 and caveolin-1 were deregulated following treatment. These observations were consistent with inhibition of signal transduction and suggested a possible mechanism of resistance or recovery from 17-allylamino-17-demethoxygeldanamycin treatment. The results shed light on the molecular mode of action of the hsp90 inhibitors, and suggest possible molecular markers of drug action for use in hypothesis testing clinical trials. Oncogene (2000) 19, 4125 - 4133.

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Apoptosis was induced by treating L1210 leukaemia cells with mechlorethamine, and SW620 colorectal cells with doxorubicin. The onset and progression of apoptosis were monitored by assessing caspase activation, mitochondrial transmembrane potential, phosphatidylserine externalization, DNA fragmentation and cell morphology. In parallel, 31P magnetic resonance (MR) spectra of cell extracts were recorded. In L1210 cells, caspase activation was detected at 4 h. By 3 h, the MR spectra showed a steady decrease in NTP and NAD, and a significant build-up of fructose 1,6-bisphosphate (F-1,6-P) dihydroxyacetonephosphate and glycerol-3-phosphate, indicating modulation of glycolysis. Treatment with iodoacetate also induced a build-up of F-1,6-P, while preincubation with two poly(ADP-ribose) polymerase inhibitors, 3-aminobenzamide and nicotinamide, prevented the drop in NAD and the build-up of glycolytic intermediates. This suggested that our results were due to inhibition of glyceraldehyde-3-phosphate dehydrogenase, possibly as a consequence of NAD depletion following poly(ADP-ribose) polymerase activation. Doxorubicin treatment of the adherent SW620 cells caused cells committed to apoptosis to detach. F-1,6-P was observed in detached cells, but not in treated cells that remained attached. This indicated that our observations were not cell line- or treatment-specific, but were correlated with the appearance of apoptotic cells following drug treatment. The 31P MR spectrum of tumours responding to chemotherapy could be modulated by similar effects..

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BACKGROUND: Kirsten ras (Ki-ras) gene mutations occur early in the progression of colorectal adenoma to carcinoma. The aim of this collaborative study was to clarify the association between Ki-ras mutations, patient outcome, and tumor characteristics by use of data from colorectal cancer patients worldwide. METHODS: Investigators who had published data on Ki-ras and colorectal cancer were invited to complete a questionnaire for each patient entered into a database. Two-sided statistical tests were used to analyze data. RESULTS: Patients (n = 2721) were recruited from 22 groups in 13 countries. Mutations of Ki-ras codon 12 (wild type = GGT = glycine) or codon 13 (wild type = GGC = glycine) were detected in 37.7% of the tumors; 80.8% (584 of 723) of all the specified mutations occurred in codon 12, and 78.1% (565 of 723) of all the specified mutations were at the second base of either codon. Mutations were not associated with sex, age, tumor site, or Dukes' stage. Mutation rates seen in patients with sporadic tumors were comparable to those observed in patients with a predisposing cause for their cancer. Poorly differentiated tumors were less frequently mutated (P = .002). Multivariate analysis suggested that the presence of a mutation increased risk of recurrence (P<.001) and death (P = .004). In particular, any mutation of guanine (G) to thymine (T) but not to adenine (A) or to cytosine (C) increased the risk of recurrence (P = .006) and death (P<.001). When individual, specific mutations were evaluated, only valine codon 12 was found to convey an independent, increased risk of recurrence (P = .007) and death (P = .004). CONCLUSIONS: Ki-ras mutations are associated with increased risk of relapse and death, but some mutations are more aggressive than others..

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A direct, ribonuclease T1 protection assay was employed to study the binding of a delta agent genomic RNA transcript containing the conserved domain to the double-stranded RNA- (dsRNA-) dependent protein kinase of mammalian cells, PKR (also known as DAI, p1-elF2, or p68 kinase). In a control reaction, this assay identified a major portion of the same PKR binding site in VA RNA as deduced previously using a footprinting technique (Clarke PA, Mathews MB, 1995, RNA 1:1-20). Although delta agent RNA contains extensive secondary structure throughout the conserved region, we found a remarkable specificity in its PKR binding. The same region was protected by intact PKR and by a 184-amino acid fragment thereof containing the two RNA-binding motifs (dsRBMs) but lacking kinase activity. Two specific opposed, continuous segments of delta agent RNA (extending about 65-70 bases) were obtained reproducibly. Each is more than twice as long as those protected in VA RNA (about 25 bases), suggesting the involvement of PKR dimers in delta RNA binding. The PKR-protected region of delta agent RNA also contains a characteristic tertiary structural element that may be involved in binding specificity..

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BACKGROUND: Published data are contradictory about the importance of K-ras mutations in advanced tumours and are not available for early cancers. AIMS: To establish whether specific K-ras mutations are prognostic markers in early stage colorectal adenocarcinoma. METHODS: The presence of K-ras exon 1 mutations were correlated with tumour recurrence in two groups of patients: group 1 was a consecutive series of patients with resected colorectal adenocarcinoma at low risk of recurrence; group 2 were patients referred for chemotherapy after relapse of previously resected early stage tumours. K-ras mutations were detected by direct sequencing of whole tissue samples in all patients and in some, the leading edge and centre of the tumour were also microdissected out individually and sequenced. RESULTS: Mutations were present in 26 (26.5%) of 98 patients in group 1; 14 patients developed a recurrence, four (28.5%) of whom had a K-ras mutation. Seventy nine patients have not developed tumour recurrence, 22 (28%) of whom had a mutation (p = 0.84). K-ras mutations were present in five of 14 patients in group 2. Microdissection did not increase the number of mutations detected. CONCLUSIONS: Individual K-ras genotypes are distributed homogeneously throughout early stage colorectal adenocarcinomas, but detection of a mutation has no apparent prognostic value..

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BACKGROUND: Overexpression of BCL-2 is common in non-Hodgkin lymphoma and leads to resistance to programmed cell death (apoptosis) and promotes tumorigenesis. Antisense oligonucleotides targeted at the open reading frame of the BCL-2 mRNA cause a specific down-regulation of BCL-2 expression which leads to increased apoptosis. Lymphoma grown in laboratory animals responds to BCL-2 antisense oligonucleotides with few toxic effects. We report the first study of BCL-2 antisense therapy in human beings. METHODS: A daily subcutaneous infusion of 18-base, fully phosporothioated antisense oligonucleotide was administered for 2 weeks to nine patients who had BCL-2-positive relapsed non-Hodgkin lymphoma. Toxicity was scored by the common toxicity criteria, and tumour response was assessed by computed tomography scan. Efficacy was also assessed by quantification of BCL-2 expression; BCL-2 protein levels were measured by flow cytometry in samples from patients. FINDINGS: During the course of the study, the daily dose of BCL-2 antisense was increased incrementally from 4.6 mg/m2 to 73.6 mg/m2. No treatment-related toxic effects occurred, apart from local inflammation at the infusion site. In two patients, computed tomography scans showed a reduction in tumour size (one minor, one complete response). In two patients, the number of circulating lymphoma cells decreased during treatment. In four patients, serum concentrations of lactate dehydrogenase fell, and in two of these patients symptoms improved. We were able to measure BCL-2 levels by flow cytometry in the samples of five patients, two of whom had reduced levels of BCL-2 protein. INTERPRETATION: In patients with relapsing non-Hodgkin lymphoma, BCL-2 antisense therapy led to an improvement in symptoms, objective biochemical and radiological evidence of tumour response, and down-regulation of the BCL-2 protein in some patients. Our findings are encouraging and warrant further investigations of BCL-2 antisense therapy in cancer treatment..

In the last year, a number of studies have reported the expression of bcl-2 in colorectal adenocarcinomas. However, the influence of bcl-2 expression on response to chemotherapy and outcome in patients with advanced colorectal adenocarcinoma has not been reported. We analysed bcl-2 expression in 231 colorectal tumours from patients that were treated by surgery alone or with 5-fluorouracil-based chemotherapy. Of 231 tumours, 149 (64.5%) overexpressed bcl-2. Bcl-2 expression was associated with low plasma CEA levels (P=0.013) and inversely associated with poor differentiation (P=0.049). However, bcl-2 expression did not significantly influence failure-free or overall survival in surgically treated patients. In the group of patients receiving 5-fluorouracil-based chemotherapy bcl-2 expression did not influence response to chemotherapy; nor did it effect failure-free or overall survival..

The Bcl-2 protein is capable of preventing apoptosis, and in vitro evidence suggests a role in drug resistance. It is expressed and the gene is rearranged in a proportion of cases of large-cell non-Hodgkin's lymphoma (NHL), but the clinical significance of these findings is controversial. The purpose of this study was to determine the influence of both Bcl-2 expression and major breakpoint region (MBR) bcl-2 rearrangement in a large cohort of prospectively accrued patients with intermediate-grade B-cell NHL treated in a standardized manner. All patients with Working Formulation F, G, or H NHL treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy in British National Lymphoma investigation studies between July 1974 and April 1992 were considered for this study if the appropriate paraffin blocks were available. Paraffin sections from the diagnostic specimen were analyzed for evidence of MBR rearrangement using a polymerase chain reaction-based method, and for Bcl-2 expression using immunohistochemistry. Failure to achieve complete remission (CR), relapse, death from NHL, and deaths from all causes were used as end points to measure CR rate, actuarial relapse rate, actuarial survival from NHL, and actuarial overall survival. One hundred sixty-one suitable patients were identified and tested for the bcl-2 MBR translocation, with 27 (17%) found to be positive; 153 of these patients were tested with immunocytochemistry, and 84 (55%) showed evidence of Bcl-2 expression. For patients who achieved CR from the initial treatment, the relapse rate was significantly higher in those with Bcl-2 expression than in those without. In addition, multivariate analysis identified Bcl-2 expression as the only factor significantly related to relapse rate in the subjects measured. The cause-specific survival for NHL in the series as a whole was significantly lower in patients with Bcl-2 expression than in those without. MBR status had no significant influence on any of the outcome measures, but the number of MBR-positive patients was relatively small, and larger studies are required. In conclusion, in Working Formulation F, G, and H NHL of B-cell type, expression of Bcl-2 protein predicted independently for relapse..

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The protein kinase DAI, the double-stranded RNA activated inhibitor of translation (also known as PKR), regulates cell growth, virus infection, and other processes. DAI represents a class of proteins containing a recently recognized RNA binding motif, the dsRBM, but little is known about the contacts between these proteins and their RNA ligands. In adenovirus-infected cells, DAI activation is prevented by VA RNAI, a highly structured RNA that binds to the kinase. VA RNA contains three chief structural features: a terminal stem, an apical stem-loop, and a complex central domain. We used enzymatic and chemical footprinting to identify the interactions between DAI and VA RNAI. DAI protects the proximal part of the apical stem structure, an adjacent region in the central domain, and a region surrounding a conserved stem in the central domain from nuclease attack. During binding the RNA undergoes a conformational change that is mainly restricted to the central domain. A similar change is induced by magnesium ions alone. Footprinting and interference binding assays using base-specific chemical probes suggest that the protein does not make major contacts with RNA bases. On the other hand, footprinting with probes specific for the RNA backbone shows that DAI engages in a strong interaction with the minor groove of the apical stem and a weaker interaction in the central domain. A truncated form of DAI, p20, containing only the RNA binding domain, gives a similar protection pattern in the apical stem but protects the central domain less effectively. We conclude that the RNA binding domain of DAI interacts directly with the apical stem and central domain of VA RNA, and that other regions of the protein contribute to interactions with the central domain..

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BHRF1 is an Epstein-Barr virus encoded protein which has a 38% sequence similarity with bcl-2 over the carboxyl portion. Like bcl-2, BHRF1 has been shown to suppress programmed cell death from apoptosis. Previously BHRF1 has been detected in mitochondrial, microsomal, and nuclear compartments by cell fractionation analysis. In this study we have used the technique of immunoelectron microscopy to define the ultrastructural distribution of the BHRF1 product in the EBV converted cell lines B95.8 and P3HR-1. The BHRF1 product was localized at the periphery of the mitochondria in a pattern similar to that of bcl-2 and by analogy with bcl-2 this is likely to be the functional destination. Sequence analysis of the BHRF1 protein disclosed similarity with the recently described bcl-2 homologues bcl-x (32%) and bax (34%) over the carboxyl portion, with several domains of complete identity. BHRF1 appears to be a member of a gene family involved in the regulation of programmed cell death. The identity between BHRF1 and bcl-2, an apparent shared ability to abrogate apoptosis, and the common ultrastructural localization is compelling and suggests that bcl-2 and BHRF1 are both functionally and mechanistically similar..

The relative levels, rates of synthesis and stabilities of the abundant Epstein-Barr virus (EBV)-encoded small RNAs, EBER-1 and EBER-2, were examined in Daudi Burkitt's lymphoma cells. Although both RNAs are transcribed at approximately equal rates, the steady-state level of EBER-1 is at least 10-fold greater than that of EBER-2. This is shown to be due to a much faster rate of turnover of EBER-2. In the presence of actinomycin D, the half-lives of EBER-1 and EBER-2 are 8 to 9 h and 0.75 h, respectively. Following treatment of the cells with human interferon (IFN) alpha the transcription of both RNAs is strongly inhibited. However, the level of EBER-1 increases up to twofold, indicating a further stabilization of this RNA. In IFN-treated cells, EBER-2 accumulates in the form of truncated products. Nuclease protection experiments indicated that this is due to a post-transcriptional modification of the 3' end of the molecule. These data show that the effects of IFN treatment on the expression of these two viral gene products are very complex in cells latently infected with EBV..

Epstein-Barr virus encodes two small RNAs, EBER-1 and -2, that are abundantly expressed in latently infected cells. Recent evidence suggests a role for EBER-1 in regulation of translation since this RNA is able to prevent the inhibition of protein synthesis by double-stranded RNA in rabbit reticulocyte lysates. We show here that EBER-1 that has been synthesized in vitro forms a complex with the dsRNA-activated inhibitor of protein synthesis DAI, a protein kinase that specifically phosphorylates polypeptide chain initiation factor eIF-2. Gel retardation assays and UV crosslinking experiments indicate that complex formation is specific for EBER-1 and requires the presence of some secondary structure in the molecule. RNA competition studies show that EBER-1-DAI complex formation is not inhibited in the presence of other small RNA species, heparin or the synthetic double-stranded RNA, poly(I).poly(C). SDS gel analysis reveals the existence of two forms of the crosslinked complex, of 64-68kDa and 46-53kDa, both of which are recognized by anti-DAI antibodies in immunoprecipitation experiments. These data suggest that EBER-1 regulates protein synthesis through its ability to interact with DAI..

A role for the Epstein-Barr virus small RNA species EBER-1 in the regulation of protein synthesis has been investigated in the reticulocyte-lysate cell-free translation system. Recombinant EBER-1 was synthesized by in vitro transcription of a plasmid containing the viral gene and purified by CF11-cellulose chromatography and ribonuclease III treatment. When added to the reticulocyte lysate at 10-20 micrograms/ml or more, EBER-1 prevents the inhibition of protein synthesis caused by low concentrations of synthetic double-stranded RNA, poly(I).poly(C). This effect is eliminated by treatment of the recombinant EBER-1 with ribonuclease T1. Disruption of the secondary structure of EBER-1 by substitution of inosine for guanosine in the in-vitro-synthesized RNA impairs the ability of EBER-1 to prevent the poly(I).poly(C)-mediated inhibition of protein synthesis. These results suggest that high concentrations of EBER-1 regulate protein synthesis by blocking the activation of the double-stranded RNA-dependent eukaryotic initiation factor 2 alpha (eIF-2 alpha) protein kinase DAI (p68), and that this property is dependent on the secondary structure of the small RNA molecule..

This paper presents data on the effects of interferon treatment on Epstein-Barr virus (EBV) gene expression in latently infected Daudi Burkitt's lymphoma cells, and reviews the possible role of viral gene products in the regulation of translation. In Daudi cells the main virally coded RNAs are the small untranslated RNAs EBER-1 and EBER-2, two mRNAs for the DNA binding protein EBNA-1, and a number of small RNAs containing sequences from the BamHI W repeat region of the viral genome. Interferon treatment does not change the qualitative pattern of EBV gene expression but decreases the levels of the EBNA-1 mRNAs. The chromatographic behaviour of EBV-encoded RNAs on CF11-cellulose indicates that many contain double-stranded regions; these RNAs co-purify with RNA that activates the interferon-induced, dsRNA-sensitive protein kinase DAI. Computer analysis indicates that the exons transcribed from the BamHI W repeats have the potential for formation of very stable secondary structures. Many viruses can counteract the inhibition of protein synthesis mediated by the DAI-catalysed phosphorylation of initiation factor eIF-2 and our data suggest that the small RNA EBER-1 may fulfil this function in the EBV system. During the infection and immortalization of B lymphocytes by EBV the synthesis of large amounts of EBER-1 RNA might thus allow the virus to circumvent one of the interferon-mediated mechanisms of host cell defence..

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Phosphatidylinositol-3-kinase (PI3K) is an important target in cancer due to the deregulation of the PI3K/ Akt signaling pathway in a wide variety of tumors. A series of thieno[3,2-d]pyrimidine derivatives were prepared and evaluated as inhibitors of PI3 kinase p110alpha. The synthesis, biological activity, and further profiling of these compounds are described. This work resulted in the discovery of 17, GDC-0941, which is a potent, selective, orally bioavailable inhibitor of PI3K and is currently being evaluated in human clinical trials for the treatment of cancer..