LSD1 inhibition suppresses ASCL1 and de-represses YAP1 to drive potent activity against neuroendocrine prostate cancer.

Lysine-specific demethylase 1 (LSD1 or KDM1A ) has emerged as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Among mCRPC subtypes, neuroendocrine prostate cancer (NEPC) is an exceptionally aggressive variant driven by lineage plasticity, an adaptive resistance mechanism to androgen receptor axis-targeted therapies. Our study shows that LSD1 expression is elevated in NEPC and associated with unfavorable clinical outcomes. Using genetic approaches, we validated the on-target effects of LSD1 inhibition across various models. We investigated the therapeutic potential of bomedemstat, an orally bioavailable, irreversible LSD1 inhibitor with low nanomolar potency. Our findings demonstrate potent antitumor activity against CRPC models, including tumor regressions in NEPC patient-derived xenografts. Mechanistically, our study uncovers that LSD1 inhibition suppresses the neuronal transcriptional program by downregulating ASCL1 through disrupting LSD1:INSM1 interactions and de-repressing YAP1 silencing. Our data support the clinical development of LSD1 inhibitors for treating CRPC - especially the aggressive NE phenotype. Statement of Significance: Neuroendocrine prostate cancer presents a clinical challenge due to the lack of effective treatments. Our research demonstrates that bomedemstat, a potent and selective LSD1 inhibitor, effectively combats neuroendocrine prostate cancer by downregulating the ASCL1- dependent NE transcriptional program and re-expressing YAP1.

Genome-wide gene expression in a pharmacological hormonal transition model and its relation to depressive symptoms.

OBJECTIVES: Sensitivity to sex-steroid hormone fluctuations may increase risk for perinatal depression. We aimed to identify genome-wide biological profiles in women demonstrating sensitivity to pharmacological sex-hormone manipulation with gonadotrophin-releasing hormone agonist (GnRHa). METHODS: Longitudinal gene expression (Illumina Human HT12.v4) and DNA methylation data (Infinium HumanMethylation450K BeadChip) from 60 women (30 GnRHa, 30 placebo) were generated (Trial ID: NCT02661789). Differences between baseline and two follow-up points (initial stimulation- and subsequent early suppression phase) in the biphasic ovarian hormone response to GnRHa were assessed using linear mixed effects models. RESULTS: Genome-wide analysis revealed 588 probes differentially expressed from GnRHa intervention to first stimulatory phase follow-up (intervention group × time) after 10% fdr multiple testing correction. Of these, 54% genes were also significantly associated with estradiol changes over time (proxy for GnRHa response magnitude), 9.5% were associated with changes in depressive symptoms, and 38% were associated with changes in neocortical serotonin transporter binding. The genes were implicated in TGF beta signaling, adipogenesis, regulation of actin cytoskeleton, and focal adhesion pathways and enriched for DNA methylation changes (P = 0.006). CONCLUSIONS: These findings point toward an altered peripheral blood transcriptomic landscape in a pharmacological model of sex-hormone-induced depressive symptoms.

Correlation of B7-H3 with androgen receptor, immune pathways and poor outcome in prostate cancer: an expression-based analysis.

BACKGROUND: B7-H3 (CD276), part of the B7 superfamily of immune checkpoint molecules, has been shown to have an immunomodulatory role. Its regulation, receptor and mechanism of action remain unclear. B7-H3 protein expression correlates with prostate cancer outcomes, and humanized monoclonal antibodies (that is, enoblituzumab) are currently being investigated for therapeutic use. Here we used genomic expression data to examine the relationship between B7-H3 mRNA expression and prostate cancer. METHODS: Prostatectomy tissue from 2781 patients were profiled using the Affymetrix HuEx 1.0 ST microarray. Pairwise comparisons were used to identify significant associations between B7-H3 expression and clinicopathologic variables, and survival analyses were used to evaluate the prognostic significance of B7-H3. Pearson's correlation analyses were performed to assess the relationship of B7-H3 expression with molecular subtypes and individual transcripts. Androgen receptor (AR) occupancy at the B7-H3 locus was determined using chromatin immunoprecipitation (ChIP), and androgen-dependent expression changes in B7-H3 was evaluated by quantitative reverse transcription PCR in LNCaP cell lines. Oncomine was queried to evaluate B7-H3 expression in metastatic disease. RESULTS: B7-H3 mRNA expression was positively associated with higher Gleason score (P<0.001), tumor stage (P<0.001), and castrate resistant metastatic disease (P<0.0001). High B7-H3 expression correlated with the development of metastasis and prostate cancer specific mortality, but this was not significant on multi-variable analysis. B7-H3 expression correlated with ERG-positive disease (r=0.99) and AR expression (r=0.36). ChIP revealed an AR-binding site upstream of B7-H3, and the presence of androgens decreased B7-H3 expression in LNCaP suggesting potential direct AR regulation. Gene set enrichment analysis demonstrated an association of B7-H3 with androgen signaling as well as immune regulatory pathways. CONCLUSIONS: Higher B7-H3 expression correlates with Gleason grade, prostate cancer stage and poor oncologic outcomes in prostatectomy cohorts. B7-H3 expression appears to be related to androgen signaling as well as the immune reactome.

History of Orphan Drug Regulation-United States and Beyond.

The US Orphan Drug Act, passed in 1982, was the first orphan drug legislation in the world. It is a law based on economic incentives making it financially possible for pharmaceutical firms to develop products for small patient populations. Since passage, many additional countries have developed orphan drug programs and many pharmaceutical firms have developed around the orphan program. Today, more than 500 drugs for rare diseases have been developed in the United States.

Early predictive biomarkers for postpartum depression point to a role for estrogen receptor signaling.

BACKGROUND: Postpartum depression (PPD) affects approximately 13% of women and has a negative impact on mother and infant, hence reliable biological tests for early detection of PPD are essential. We aimed to identify robust predictive biomarkers for PPD using peripheral blood gene expression profiles in a hypothesis-free genome-wide study in a high-risk, longitudinal cohort. METHOD: We performed a genome-wide association study in a longitudinal discovery cohort comprising 62 women with psychopathology. Gene expression and hormones were measured in the first and third pregnancy trimesters and early postpartum (201 samples). The replication cohort comprised 24 women with third pregnancy trimester gene expression measures. Gene expression was measured on Illumina-Human HT12 v4 microarrays. Plasma estradiol and estriol were measured. Statistical analysis was performed in R. RESULTS: We identified 116 transcripts differentially expressed between the PPD and euthymic women during the third trimester that allowed prediction of PPD with an accuracy of 88% in both discovery and replication cohorts. Within these transcripts, significant enrichment of transcripts implicated that estrogen signaling was observed and such enrichment was also evident when analysing published gene expression data predicting PPD from a non-risk cohort. While plasma estrogen levels were not different across groups, women with PPD displayed an increased sensitivity to estrogen signaling, confirming the previously proposed hypothesis of increased sex-steroid sensitivity as a susceptibility factor for PPD. CONCLUSIONS: These results suggest that PPD can be robustly predicted in currently euthymic women as early as the third trimester and these findings have implications for predictive testing of high-risk women and prevention and treatment for PPD.

Assessing the order of critical alterations in prostate cancer development and progression by IHC: further evidence that PTEN loss occurs subsequent to ERG gene fusion.

BACKGROUND: ERG rearrangements and PTEN (phosphatase and tensin homolog deleted on chromosome 10) loss are two of the most common genetic alterations in prostate cancer. However, there is still significant controversy regarding the order of events of these two changes during the carcinogenic process. We used immunohistochemistry (IHC) to determine ERG and PTEN status, and calculated the fraction of cases with homogeneous/heterogeneous ERG and PTEN staining in a given tumor. METHODS: Using a single standard tissue section from the index tumor from radical prostatectomies (N=77), enriched for relatively high grade and stage tumors, we examined ERG and PTEN status by IHC. We determined whether ERG or PTEN staining was homogeneous (all tumor cells staining positive) or heterogeneous (focal tumor cell staining) in a given tumor focus. RESULTS: Fifty-seven percent (N=44/77) of tumor foci showed ERG positivity, with 93% of these (N=41/44) cases showing homogeneous ERG staining in which all tumor cells stained positively. Fifty-three percent (N=41/77) of tumor foci showed PTEN loss, and of these 66% (N=27/41) showed heterogeneous PTEN loss. In ERG homogeneously positive cases, any PTEN loss occurred in 56% (N=23/41) of cases, and of these 65% (N=15/23) showed heterogeneous loss. In ERG-negative tumors, 51.5% (N=17/33) showed PTEN loss, and of these 64.7% (N=11/17) showed heterogeneous PTEN loss. In a subset of cases, genomic deletions of PTEN were verified by fluorescence in situ hybridization in regions with PTEN protein loss as compared with regions with intact PTEN protein, which did not show PTEN genomic loss. CONCLUSIONS: These results support the concept that PTEN loss tends to occur as a subclonal event within a given established prostatic carcinoma clone after ERG gene fusion. The combination of ERG and PTEN IHC staining can be used as a simple test to ascertain PTEN and ERG gene rearrangement status within a given prostate cancer in either a research or clinical setting.

Cold-atom scanning probe microscopy.

Scanning probe microscopes are widely used to study surfaces with atomic resolution in many areas of nanoscience. Ultracold atomic gases trapped in electromagnetic potentials can be used to study electromagnetic interactions between the atoms and nearby surfaces in chip-based systems. Here we demonstrate a new type of scanning probe microscope that combines these two areas of research by using an ultracold gas as the tip in a scanning probe microscope. This cold-atom scanning probe microscope offers a large scanning volume, an ultrasoft tip of well-defined shape and high purity, and sensitivity to electromagnetic forces (including dispersion forces near nanostructured surfaces). We use the cold-atom scanning probe microscope to non-destructively measure the position and height of carbon nanotube structures and individual free-standing nanotubes. Cooling the atoms in the gas to form a Bose-Einstein condensate increases the resolution of the device.

Tailoring gold nanostructures for near-field optical applications.

A method of combined thin-film deposition, electron beam lithography, and ion milling is presented for the fabrication of gold and silver nanostructures. The flexibility of lithographical processes for the variation of geometric parameters is combined with three-dimensional control over the surface evolution. Depending on the etching angle, different shapes ranging from cones over rods to cups can be achieved. These size- and shape-tunable structures present a toolbox for nano-optical investigations. As an example, optical properties of systematically varying structures are examined in a parabolic mirror confocal microscope.

Characterization of the morphology and composition of commercial negative resists used for lithographic processes.

We present a spectroscopic and microscopic characterization of the chemical composition, structure, and morphology of two commercial negative resists using Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). For this purpose, films of a novolak-based resist (ma-N 2400) and hydrogen silsesquioxane (HSQ) are treated under different conditions (temperature, deep ultraviolet (DUV) exposure, CHF(3) plasma). Topographic AFM images show that both heating and DUV exposure strongly affect the surface morphology of as-prepared ma-N 2400 resist films. These different treatment conditions also lead to decreasing roughnesses, which indicates structural reorganization. Furthermore, the decrease of the photoactive compound (bisazide) in the ma-N 2400 resist films, observed in FTIR spectra, suggests cross-linking of the resist after CHF(3) plasma treatment, heating, or DUV exposure. XPS measurements on different CHF(3) plasma-treated surfaces reveal that a structurally homogeneous fluorine-containing polymer is generated that is responsible for an enhanced etch resistance. FTIR measurements of HSQ films show a correlation between the degree of HSQ cross-linking and baking time.

Bovine placental steroid sulphatase: molecular cloning and expression pattern in placentomes during gestation and at parturition.

Apart from during the prepartal period, the main oestrogen produced by the bovine trophoblast is oestrone sulphate (E1S) which does not bind to nuclear oestrogen receptors (ER). High steroid sulphatase (StS) activities previously detected in the maternal part of bovine placentomes (caruncles) suggest the local activation of E1S ("sulphatase pathway"). Consequently, the expression pattern of StS in bovine placentomes was investigated by immunohistochemistry using an antiserum against human placental StS. Cross-reactivity for bovine StS was confirmed by Western blot yielding a single band of 62 kDa in both bovine and human placenta. Immunostaining for StS was detected in caruncular epithelial cells (CEC), which was clearly related to gestational age. In animals pregnant between 100 and 284 days (n=17), signals were restricted to CEC adjacent to the chorionic plate and basal primary and secondary chorionic villi. After the onset of prepartal luteolysis (days 273-282; n=3) and during active labour (n=5) overall staining intensity had increased substantially and signals occurred ubiquitously in the flattened and partially dismantled caruncular epithelium. A 2204 bp full-length mRNA transcript of the bovine StS exhibiting 74% and 77% sequence identity to human StS on the mRNA and protein levels, respectively, was cloned by RACE-PCR. Real-time RT-PCR detected a 2.5-fold increase of StS-mRNA in prepartal placentomes, which, however, was not statistically significant. The co-localisation of ERalpha and StS in CEC is consistent with a role of placental oestrogens as regulators of caruncular growth and differentiation, and the up-regulation of carunclar StS may be involved in the marked prepartal increase of free oestrogens.

Orphan drug product regulation--United States.

The legislative history of the United States Orphan Drug Act began with rare, unanimous approval by the United States Congress. The Act, mid consequently the Regulations, have evolved since then. The two-stage process of Orphan Drug designation and approval is outlined, as well as the incentives that are offered to commercial companies for their implementation. Orphan Drugs are likely to be over-represented among drugs used under "Treatment" INDs. For patent- and "drug-difference" reasons, the benefits under the Orphan Drug Act are especially valuable to those who develop biologics. By any measure, this legislation, which requires only voluntary participation, has been a success; because the human genome is likely to lead to more biologicals than orthodox drugs, this success is likely to continue into the future. But even so, the 18-year experience with Orphan Drugs in the United States has led to some 225 Orphan Product approvals that benefit many millions of patients.

Trafficking of endothelial nitric-oxide synthase in living cells. Quantitative evidence supporting the role of palmitoylation as a kinetic trapping mechanism limiting membrane diffusion.

To examine endothelial nitric-oxide synthase (eNOS) trafficking in living endothelial cells, the eNOS-deficient endothelial cell line ECV304 was stably transfected with an eNOS-green fluorescent protein (GFP) fusion construct and characterized by functional, biochemical, and microscopic analysis. eNOS-GFP was colocalized with Golgi and plasma membrane markers and produced NO in response to agonist challenge. Localization in the plasma membrane was dependent on the palmitoylation state, since the palmitoylation mutant of eNOS (C15S/C26S eNOS-GFP) was excluded from the plasma membrane and was concentrated in a diffuse perinuclear pattern. Fluorescence recovery after photobleaching (FRAP) revealed eNOS-GFP in the perinuclear region moving 3 times faster than the plasmalemmal pool, suggesting that protein-lipid or protein-protein interactions are different in these two cellular domains. FRAP of the palmitoylation mutant was two times faster than that of wild-type eNOS-GFP, indicating that palmitoylation was influencing the rate of trafficking. Interestingly, FRAP of C15S/C26S eNOS-GFP but not wild-type eNOS-GFP fit a model of protein diffusion in a lipid bilayer. These data suggest that the regulation of eNOS trafficking within the plasma membrane and Golgi are probably different mechanisms and not due to simple diffusion of the protein in a lipid bilayer.

School-based methylphenidate placebo protocols: methodological and practical issues.

Around 1990, psychologists and educators began to notice increasing use of methylphenidate by students. Diagnosis of attention-deficit/hyperactivity disorder by family physicians and pediatricians was most commonly based on brief behavioral descriptions by parents and, infrequently, by use of rating scales. At that time, the present researchers began to explore the development of a school-based, methodologically sound, and inexpensive method of assessing the efficacy of stimulant medications, which would ensure reasonable compliance by teachers, parents, and students in monitoring the effects of medications and placebos. This article focuses on the methodological issues involved in choosing instruments to monitor behavior, once a comprehensive evaluation has suggested trials on Ritalin. Case examples illustrate problems of teacher compliance in filling out measures, supplying adequate placebos, and obtaining physician cooperation, and with the practical issue of providing adequate data without overwhelming the time and resources of participants. Emerging school-based methodologies are discussed with recommendations for future efforts.

The size of sebaceous glands in relation to the size of hair follicles on the heads of some small mammals (Insectivora, Chiroptera, Rodentia).

Many large sebaceous glands have been described, and functions such as scent production suggested, but gland size has seldom been studied in relation to the surface area of their hair follicles. However, investigating this relationship is essential for establishing whether glands produce more sebum than would be required for lubricating associated hair alone. Here, the relationship between sebaceous gland size and the surface area of the associated hair follicles has been studied. Glands on the heads of 20 species from three orders were compared with those associated with fur hairs. In all species, the fur hairs had small hair follicles with small sebaceous glands, as had the mystacial and submandibular hairs of Rodentia. Medium-sized glands and hairs were found in the mystacial and submandibular region in Insectivora, and in the circumoral region in Chiroptera and Rodentia. Large glands and hairs were found on a pad in the corner of the mouth in Rodentia. Although the size of glands was not directly proportional to hair size, some gross trends were noted. Vibrissae had either no or very small sebaceous glands. It is likely that sebum has to be provided from elsewhere to lubricate their surfaces. The glands on the snout of Insectivora and Chiroptera are clearly enlarged and could probably produce more sebum than would be required for grooming vibrissae alone. In Rodentia, vibrissae were surrounded by small hair and glands, and the nearest glands large enough to provide sebum were the glands on the pad in the mouth corner.

A tendon-locking mechanism in two climbing rodents, Muscardinus avellanarius and Micromys minutus (Mammalia, Rodentia).

Having compared the microanatomy of the toes of a terrestrial to two climbing species, adaptations were found in the flexor tendons and in the integument. In contrast to Crocidura russula, both Muscardinus avellanarius and Micromys minutus have a tendon-locking mechanism (TLM) that is engaged when the middle phalanx is bent. A ventral thickening of the flexor tendon is situated deep to a thickened portion of the ventral tendon sheath. When twigs or stalks are grasped, the TLM allows less muscular energy to be expended. In C. russula glands are restricted to the terminal pads, but in the climbing species they occur in the sole of the toes as well. In the reed-living M. minutus knob-shaped integumental thickenings, together with the digital pads, stabilize the grip. In contrast the arboreal M. avellanarius often climbs thick branches and shows adaptations for pressing the sole of the feet against the surface. Thereby the tendon attached to the plantar integument of the toes transfers the muscle force directly to the bark. Unlike the other digits on the forefeet of both climbing species, no TLM is present in the anterior digit. In M. minutus this short digit is twisted towards the palm and, with the carpal pads, provides an abutment against the grasping fingers. In M. avellanarius the anterior digit has very thin tendons and is that much reduced in length that it is completely integrated into the digital pad where it acts, at best, as a lateral support of the pad.

Evaluation of orphan products by the U.S. Food and Drug Administration.

Orphan drug products generally are used in treating or preventing rare diseases. The small number of patients available for study may create special problems in the evaluation of these products. This paper examines some of the special problems that are associated with the design and implementation of studies to evaluate the safety and efficacy of orphan drugs. The U.S. Food and Drug Administration (FDA) has not established special criteria for evaluating orphan drugs per se, but the FDA has been flexible in evaluating drug products that present special problems, especially when these products are for treatment of serious of life-threatening illnesses. The FDA and other U.S. governmental agencies also have taken steps to promote the development and availability of drugs for rare diseases, including making these products available to patients who are in need, even before the drugs have full FDA marketing approval.