Phase I study of onapristone, a type I antiprogestin, in female patients with previously treated recurrent or metastatic progesterone receptor-expressing cancers.

INTRODUCTION: Onapristone is a type I progesterone receptor (PR) antagonist, which prevents PR- mediated DNA transcription. Onapristone is active in multiple preclinical models and two prior studies demonstrated promising activity in patients with breast cancer. We conducted a study of extended release (ER) Onapristone to determine a recommended dose and explore the role of transcriptionally-activated PR (APR), detected as an aggregated subnuclear distribution pattern, as a predictive biomarker. METHODS: An open-label, multicenter, randomized, parallel-group, phase 1 study (target n = 60; NCT02052128) included female patients ≥18 years with PRpos tumors. APR analysis was performed on archival tumor tissue. Patients were randomized to five cohorts of extended release (ER) onapristone tablets 10, 20, 30, 40 or 50 mg BID, or immediate release 100 mg QD until progressive disease or intolerability. Primary endpoint was to identify the recommended phase 2 dose. Secondary endpoints included safety, clinical benefit and pharmacokinetics. RESULTS: The phase 1 dose escalation component of the study is complete (n = 52). Tumor diagnosis included: endometrial carcinoma 12; breast cancer 20; ovarian cancer 13; other 7. Median age was 64 (36-84). No dose limiting toxicity was observed with reported liver function test elevation related only to liver metastases. The RP2D was 50 mg ER BID. Median therapy duration was 8 weeks (range 2-44), and 9 patients had clinical benefit ≥24 weeks, including 2 patients with APRpos endometrial carcinoma. CONCLUSION: Clinical benefit with excellent tolerance was seen in heavily pretreated patients with endometrial, ovarian and breast cancer. The data support the development of Onapristone in endometrial endometrioid cancer. Onapristone should also be evaluated in ovarian and breast cancers along with APR immunohistochemistry validation.

Tumour and cellular distribution of activated forms of PR in breast cancers: a novel immunohistochemical analysis of a large clinical cohort.

BACKGROUND: The progesterone receptor (PR) is expressed by ∼70% of early breast tumours and is implicated in the progression of breast cancer. In cancerous tissues PR may be activated in the absence of a ligand, or when ligand concentrations are very low, resulting in aberrantly activated PR (APR). The presence of APR may indicate that patients with breast cancer are more likely to respond to antiprogestins. The aims of this study were to describe and classify the histological subnuclear morphology of active and inactive PR in archival breast cancer samples. METHODS: Archived tumour specimens from 801 women with invasive breast cancer were collected. Tissue samples (n=789) were analysed for PR isoforms A and B (PRA and PRB), Ki67 and estrogen receptors (ERα) status, using immunohistochemistry. Medical records were used to determine human epidermal growth factor 2 (HER2) status, tumour stage and grade. RESULTS: A total of 79% of tumours stained positive for either PRA or PRB, and of these 25% of PRA-positive and 23% of PRB-positive tumours had PR present in the activated form. APRA was associated with higher tumour grade (p=0.001). APRB was associated with a higher tumour grade (p=0.046) and a trend for a more advanced stage. Patients with PR-positive tumours treated with antiestrogens had better disease-free survival (DFS) than those with PR-negative tumours (p<0.0001). Cumulative progression rate and DFS were similar irrespective of APR status. Both APRA and APRB were independent of HER2, ERα and Ki67 expression. CONCLUSIONS: APR had a binary mode of expression in the breast cancer specimens tested, allowing separation into two tumour subsets. APR is an independent target at the cellular and tumour level and may therefore be a suitable predictive marker for antiprogestins, such as onapristone. Using the described technique, a companion diagnostic is under development to identify APR in solid tumours.

A novel, blocking, Fc-silent anti-CD40 monoclonal antibody prolongs nonhuman primate renal allograft survival in the absence of B cell depletion.

CD40-CD154 pathway blockade prolongs renal allograft survival in nonhuman primates (NHPs). However, antibodies targeting CD154 were associated with an increased incidence of thromboembolic complications. Antibodies targeting CD40 prolong renal allograft survival in NHPs without thromboembolic events but with accompanying B cell depletion, raising the question of the relative contribution of B cell depletion to the efficacy of anti-CD40 blockade. Here, we investigated whether fully silencing Fc effector functions of an anti-CD40 antibody can still promote graft survival. The parent anti-CD40 monoclonal antibody HCD122 prolonged allograft survival in MHC-mismatched cynomolgus monkey renal allograft transplantation (52, 22, and 24 days) with accompanying B cell depletion. Fc-silencing yielded CFZ533, an antibody incapable of B cell depletion but still able to potently inhibit CD40 pathway activation. CFZ533 prolonged allograft survival and function up to a defined protocol endpoint of 98-100 days (100, 100, 100, 98, and 76 days) in the absence of B cell depletion and preservation of good histological graft morphology. CFZ533 was well-tolerated, with no evidence of thromboembolic events or CD40 pathway activation and suppressed a gene signature associated with acute rejection. Thus, use of the Fc-silent anti-CD40 antibody CFZ533 appears to be an attractive approach for preventing solid organ transplant rejection.

Development of a technique to detect the activated form of the progesterone receptor and correlation with clinical and histopathological characteristics of endometrioid adenocarcinoma of the uterine corpus.

OBJECTIVE: Hormonal therapy is generally reserved for patients with endometrial cancers that fail cytotoxic chemotherapy, but there is a lack of sufficiently sensitive diagnostics to identify potential responders. We sought to develop a diagnostic technique to detect activated progesterone receptors (APR) in endometrial cancers using routine immunohistochemistry (IHC) and to correlate the presence of APR with other histopathological features and clinical disease stage. METHODS: Seventy-two tumor block specimens from patients with endometrial cancer were processed with conventional IHC methods for estrogen receptor-α (ERα), progesterone receptor (PR) and Ki67, a marker of proliferation. Tumor specimens were analyzed for the PR nuclear distribution patterns in individual tumor cells: APR positive (APR(pos)) tumors were prospectively defined as any tumor with >5% countable malignant cells with an aggregated nuclear pattern. Tumor APR status was analyzed against other biomarkers including ERα expression, Ki67 and tumor grade. RESULTS: Fifty-six of 72 samples were endometrioid. Twenty-six of 49 PR-positive endometrioid tumors (53%; 95% CI 39-67%) were APR(pos). Percent of ER(pos) cells correlated with % PR(pos) malignant cells (p=0.001, rho=0.44). APR positivity did not correlate with % PR(pos) cells in a given tumor, nor did it correlate with % Ki67 positivity; APR positivity was independent of disease stage and tumor grade (p=NS). CONCLUSIONS: In this study, approximately half of endometrioid tumors were APR(pos). APR is independent of histopathological and other known risk factors. Refining conventional PR detection has the potential to prospectively identify patients with endometrial cancer who may benefit from anti-progestin therapy.

Identification of the enzymatic active site of tobacco caffeoyl-coenzyme A O-methyltransferase by site-directed mutagenesis.

Animal catechol O-methyltransferases and plant caffeoyl-coenzyme A O-methyltransferases share about 20% sequence identity and display common structural features. The crystallographic structure of rat liver catechol O-methyltransferase was used as a template to construct a homology model for tobacco caffeoyl-coenzyme A O-methyltransferase. Integrating substrate specificity data, the three-dimensional model identified several amino acid residues putatively involved in substrate binding. These residues were mutated by a polymerase chain reaction method and wild-type and mutant enzymes were each expressed in Escherichia coli and purified. Substitution of Arg-220 with Thr resulted in the total loss of enzyme activity, thus indicating that Arg-220 is involved in the electrostatic interaction with the coenzyme A moiety of the substrate. Changes of Asp-58 to Ala and Gln-61 to Ser were shown to increase K(m) values for caffeoyl coenzyme A and to decrease catalytic activity. Deletions of two amino acid sequences specific for plant enzymes abolished activity. The secondary structures of the mutants, as measured by circular dichroism, were essentially unperturbed as compared with the wild type. Similar changes in circular dichroism spectra were observed after addition of caffeoyl coenzyme A to the wild-type enzyme and the substitution mutants but not in the case of deletion mutants, thus revealing the importance of these sequences in substrate-enzyme interactions.

Molecular modelling and endoplasmic reticulum retention of mutated TCR/CD3 complexes.

T cell receptor (TCR)/CD3 complex assembly takes place in the endoplasmic reticulum (ER). Normal TCR/CD3 complexes egress from the ER to the cis-Golgi, where the interaction with zeta2 homodimers occurs. This interaction leads to further uncontrolled transport of TCR/CD3/zeta molecules to the cell surface. The purpose of the present experiments was to determine firstly the basis for the impact of the Phe195/216 --> Val mutations on TCR/CD3 expression in Jurkat cells, and secondly why mutated J79-cell TCRalphabeta/CD3 hexamers are prevented from interacting with zeta2 homodimers. We found that Phe --> Val mutations cause serious perturbations in a so far undefined hydrophobic area formed by the two Phe195/216 on beta-strand F and aromatic/large hydrophobic amino acids on neighboring beta-strands B and A in Calpha and Cbeta domains, respectively. In addition, TCR/CD3 hexamers and zeta2 homodimers colocalize in normal Jurkat T cells, in revertant J79r58 cells, and in J79 cells transfected with wild-type TCRalpha cDNA but not in J79 mutant cells (confocal microscopy). Furthermore, mutated TCR/CD3 complexes seem to be actively retained in the ER in J79 cells but not in revertant J79r58 cells by a nondominant mechanism. We propose that a hitherto undefined ER-retention molecule controls both the protein structure and egress of TCR/CD3 complexes from the ER of alphabeta and gammadelta T cells.

The Gal/GalNAc-specific lectin from the plant pathogenic basidiomycete Rhizoctonia solani is a member of the ricin-B family.

The lectin isolated from the phytopathogenic basidiomycete Rhizoctonia solani (RSA) is a homodimer of two noncovalently associated monomers of 15.5 kDa. RSA is a basic protein (pI > 9) which consists mainly of beta-sheets. A presumed relationship with ricin-B is supported by the sequence similarity between the N-terminus of RSA and the N-terminal subdomain of ricin-B. Hydrophobic cluster analysis confirms that the N-terminus of both proteins has a comparable folding. RSA exhibits specificity towards Gal/GalNAc whereby the hydroxyls at the C3', C4', and C6' positions of the pyranose ring play a key role in the interaction with simple sugars. The carbohydrate-binding site of RSA apparently accommodates only a single sugar unit. Our results demonstrate an obvious evolutionary relationship between some fungal and plant lectins, but also provide evidence for the occurrence of a lectin consisting of subunits corresponding to a single subdomain of ricin-B.

Reactivity at the interface of chiral amphiphilic dendrimers. High asymmetric reduction by NaBH(4) of various prochiral ketones.

New amphiphilic dendrimers derived from PAMAM and D-gluconolactone were found to induce chirality in the reduction of prochiral ketones by NaBH(4), in heterogeneous (THF) and homogeneous (water) conditions. The third generation of these amphiphilic dendrimers, G(3)G, was found to be a good chiral ligand for the reduction of various prochiral ketones in heterogeneous conditions. Even with substrates well-known to give poor results (especially linear ketones), good enantioselectivities were obtained. It is also important to notice that under heterogeneous conditions (THF) the dendrimer could be recovered by filtration, regenerated, and recycled (up to 10 times), leading to reproducible results in asymmetric reduction of ketones. We have also discussed the reduction of acetophenone in water. Evidence is presented that the selectivity is dominated by the architecture of the dendrimer and some supramolecular ordering in the position of the ketone at the chiral solvating interface. The results obtained showed a correlation between stereoselectivity of the reduction and the compact character of the dendritic particles.

RNA-binding strategies common to cold-shock domain- and RNA recognition motif-containing proteins.

Numerous RNA-binding proteins have modular structures, comprising one or several copies of a selective RNA-binding domain generally coupled to an auxiliary domain that binds RNA non-specifically. We have built and compared homology-based models of the cold-shock domain (CSD) of the Xenopus protein, FRGY2, and of the third RNA recognition motif (RRM) of the ubiquitous nucleolar protein, nucleolin. Our model of the CSD(FRG)-RNA complex constitutes the first prediction of the three-dimensional structure of a CSD-RNA complex and is consistent with the hypothesis of a convergent evolution of CSD and RRM towards a related single-stranded RNA-binding surface. Circular dichroism spectroscopy studies have revealed that these RNA-binding domains are capable of orchestrating similar types of RNA conformational change. Our results further show that the respective auxiliary domains, despite their lack of sequence homology, are functionally equivalent and indispensable for modulating the properties of the specific RNA-binding domains. A comparative analysis of FRGY2 and nucleolin C-terminal domains has revealed common structural features representing the signature of a particular type of auxiliary domain, which has co-evolved with the CSD and the RRM.

Atypical binding of the neuronal POU protein N-Oct3 to noncanonical DNA targets. Implications for heterodimerization with HNF-3 beta.

The capacity of POU proteins to recognize different DNA sequences and to bind target DNA in the form of monomers, cooperative dimers or heterodimers is important in relation to their transcriptional regulatory properties. The N-Oct3 neuron-specific protein binds to an octamer-like sequence (AAATAATGC) within the (-102/-72) neuronal promoter region of the human aromatic L-amino acid decarboxylase (AADC) gene. In this atypical case the POUh and POUs tetrameric subsites are spaced one nucleotide apart and in switched order as compared with the consensus octamer. Moreover this POU binding motif overlaps the hepatocyte nuclear factor HNF-3 beta binding site (TGCTCAGTAAA) which itself contains a heptamer-like sequence (CTCAGTA). Using the isolated DNA binding domains (DBD) of the two proteins, it is shown that, when binding to this unusual recognition sequence, N-Oct3 either exhibits noncooperative homodimerization or allows the simultaneous binding of the second transcription activator HNF-3 beta. CD studies indicate that the binding of N-Oct3 monomers/dimers and N-Oct3-HNF-3 beta heterodimers to the DNA induces conformational changes of both protein and DNA. Partial proteolysis/MALDI-MS was used in conjunction with molecular modelling to show that the protein conformational change resulting from binary N-Oct3/DNA complex formation occurs within the linker peptide joining the POUs and POUh subdomains. Furthermore, modelling the N-Oct3/HNF-3 beta/DNA ternary complex predicts a nucleotide rearrangement in the overlap region and an interaction between both transcription factors. In the light of our findings, which illustrate both site-dependent and site-independent protein and DNA conformational changes, general implications for the allosteric function of DNA response elements in transcriptional regulation are discussed.

Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif.

Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.

Analysis of individual biochemical events based on artificial synapses using ultramicroelectrodes: cellular oxidative burst.

Carbon fiber platinized ultramicroelectrodes placed within micrometres of a single living cell are used to monitor cellular events. This artificial synapse is used here to collect and examine the very nature of the massive oxidative bursts produced by human fibroblasts when their membrane is locally depolarized by a puncture made with a micrometre sized sealed pipette. The electrochemical analysis of the response indicates that oxidative bursts consist of a mixture of a few femtomoles of highly cytotoxic chemicals: hydrogen peroxide, nitrogen monoxide and peroxynitrite, together with nitrite ions, which may result from a partial spontaneous decomposition of peroxynitrite prior to its release by the cell.

Distinct domains in herpes simplex virus type 1 US11 protein mediate post-transcriptional transactivation of human T-lymphotropic virus type I envelope glycoprotein gene expression and specific binding to the Rex responsive element.

Herpes simplex virus type 1 (HSV- 1) US11 protein is an RNA-binding protein which is able to mediate post-transcriptional transactivation of human T-lymphotropic virus type I (HTLV-I) envelope glycoprotein gene expression by interacting with the Rex responsive element (XRE) located at the 3' end of the env mRNA. In view of this functional activity, and because US11 protein is capable of substituting for HTLV-I Rex protein, it was hypothesized that US11 protein should exhibit at least two functional domains, an RNA-binding domain for specific interaction with the target RNA, and an effector domain involved in transport and translation of this mRNA. Recombinant US11 wild-type and deleted proteins were tested for their ability (i) to bind to the XRE and to HSV-1 UL34 RNA, the natural target of US11 protein, and (ii) to transactivate HTLV-I env gene expression. The C-terminal half of US11 protein, consisting of 20-24 XPR repeats, was necessary and sufficient to mediate RNA-binding with a high affinity and specificity. Structure prediction analyses showed the likely conformation of this domain to be that of a polyproline type II helix. Localized within the first 40 amino acids of the N-terminal region of US11 protein was the effector domain, deletion of which created US11(delta1-40), a trans-dominant negative mutant. These results demonstrate structural differences between US11 protein and proteins like Rex and Rev, despite their functional similarities.

An Arg/Lys-rich core peptide mimics TRBP binding to the HIV-1 TAR RNA upper-stem/loop.

TRBP is a cellular protein that binds to the HIV-1 leader RNA, TAR. Circular dichroism experiments have shown that a 24 amino acid peptide (TR1), located within a dsRNA binding domain (dsRBD) of TRBP, binds TAR with a 3:1 stoichiometry, eliciting a conformational change involving base unstacking. The binding characteristics of synthetic structural variants of TAR indicate that guanine residues play a key role in the TR1-RNA interaction and that binding sites exist in the upper-stem/loop and lower stem region of TAR. Deletion analysis of TR1 has led to the identification of a 15 amino acid subpeptide (TR13) which is necessary and sufficient to bind to the high affinity upper-stem/loop binding site of TAR. Alanine scanning of TR13 has revealed that mutations in either Lys or Arg residues result in altered TAR-binding, and molecular modelling/docking experiments have shown that the two Arg residues of TR13 can interact with two appropriately spaced guanine residues in the upper-stem/loop of TAR. The TR13 lysine residues appear to be essential for maintaining structural integrity and the correct positioning of the Arg side-chains. We propose that TRBP binds TAR by means of a "2-G hook" motif and that the binding specificity of this particular member of the family of double-stranded RNA-binding proteins lies within the highly conserved dsRBD core motif. Finally, our results also suggest that TRBP may function in vivo by modifying the tertiary structure of TAR RNA.

MtENOD16 and 20 are members of a family of phytocyanin-related early nodulins.

We have identified two single-copy genes from the model legume. Medicago truncatula (MtENOD16 and 20) whose expression can be correlated with early stages of root nodulation and whose predicted coding sequences are partially homologous to both pea/vetch ENOD5 and soybean N315/ENOD55. Database searching and sequence alignment have defined the encoded early nodulins as a distinct sub-family of phytocyanin-related proteins, although the absence of key ligands implies that they are unlikely to bind copper. Molecular modelling based on known phytocyanin structure has been used to predict the 3-dimensional conformation of the principle globular domain of MtENOD16/20. Additional structural features common to both early nodulin and phytocyanin precursors include an N-terminal transit peptide, a highly variable (hydroxy)proline-rich sequence which probably undergoes extensive post-translational modification, and a hydrophobic C-terminal tail.

RNA recognition by the joint action of two nucleolin RNA-binding domains: genetic analysis and structural modeling.

The interaction of nucleolin with a short stem-loop structure (NRE) requires two contiguous RNA-binding domains (RBD 1+2). The structural basis for RNA recognition by these RBDs was studied using a genetic system in Escherichia coli. Within each of the two domains, we identified several mutations that severely impair interaction with the RNA target. Mutations that alter RNA-binding specificity were also isolated, suggesting the identity of specific contacts between RBD 1+2 amino acids and nucleotides within the NRE stem-loop. Our data indicate that both RBDs participate in a joint interaction with the NRE and that each domain uses a different surface to contact the RNA. The constraints provided by these genetic data and previous mutational studies have enabled us to propose a three-dimensional model of nucleolin RBD 1+2 bound to the NRE stem-loop.

A homeodomain point mutation of the Drosophila proboscipedia protein provokes eye loss independently of homeotic function.

The Drosophila homeotic gene proboscipedia (pb: HoxA2/B2 homolog) is required for adult mouthparts development. Ectopic PB protein expression from a transgenic Hsp70-pb minigene (HSPB) results in transformation of adult antennae to maxillary palps. In contrast, most tissues appear refractory to PB-induced effects. To study the basis of homeotic tissue specificity we are isolating and studying mutations that modify dominant HSPB-induced phenotypes. One HSPB point mutation (Arg5 of the homeodomain to His) removes homeotic activity in the mouthparts and antennae, but provokes a dose-sensitive eye loss. We show that eye loss can be induced by PB proteins that no longer effectively bind to DNA. The dose-sensitive eye loss thus appears to be mediated by specific, context-dependent protein-protein interactions.

Two RNA-binding domains determine the RNA-binding specificity of nucleolin.

Nucleolin is an abundant nucleolar RNA-binding protein that seems to be involved in many aspects of ribosome biogenesis. Nucleolin contains four copies of a consensus RNA-binding domain (CS-RBD) found in several other proteins. In vitro RNA-binding studies previously determined that nucleolin interacts specifically with a short RNA stem-loop structure. Taken individually, none of the four CS-RBDs interacts significantly with the RNA target, but a peptide that contains the first two adjacent CS-RBDs (R12) is sufficient to account for nucleolin RNA-binding specificity and affinity. The full integrity of these two domains is required, since N- or C-terminal deletion abolishes the specific interaction with the RNA. Mutation of conserved amino acids within the RNP-1 sequence of CS-RBD 1 or 2 drastically reduces the interaction with the RNA, whereas mutation of the analogous residues in CS-RBDs 3 and 4 has no effect in the context of the R1234G protein (which corresponds to the C-terminal end of nucleolin). Our results demonstrate that nucleolin RNA-binding specificity is the result of a cooperation between two CS-RBDs (RBDs 1 and 2) and also suggests a direct or indirect involvement of the RNP-1 consensus sequence of both CS-RBDs in the recognition of the RNA target.

Concerted activities of the RNA recognition and the glycine-rich C-terminal domains of nucleolin are required for efficient complex formation with pre-ribosomal RNA.

Nucleolin is an abundant nucleolar protein which is involved in the early stages of ribosome assembly. The central 40-kDa domain of nucleolin comprises four RNA recognition motifs (RRM) which are presumed to be involved in specific interactions with pre-rRNA. In order to examine in detail the role of this central domain and the contribution of the N-terminal and C-terminal domains of nucleolin to RNA binding, we have used an Escherichia coli expression system to synthezise polypeptides corresponding to various combinations of the three domains and their subdomains. By means of an in-vitro binding assay and a synthetic RNA corresponding to a specific recognition site in pre-rRNA we have been able to demonstrate conclusively that the central 40-kDa domain is indeed responsible for the specificity of RNA recognition and that the N-terminal domain can be removed without affecting RNA binding. Most interestingly, it appears that the C-terminal 10-kDa domain, which is rich in glycine and arginine residues, is essential for efficient binding of nucleolin to RNA, but does not itself contribute to the specificity of the interaction. Circular dichroic spectroscopic probing of the RNA component shows that the C-terminal domain significantly modifies the RNA-binding properties of the central RRM core. Finally, infrared spectroscopic studies reveal that the central 40-kDa domain is structured in alpha helices and beta sheets and that the interaction with the specific pre-rRNA site induces subtle changes in the beta sheet conformation.