The kisspeptin receptor, crucial for hypothalamic control of puberty and reproduction, is also present in the pituitary gland. Its role in the pituitary gland is not defined. Kisspeptin signaling via the Kiss1r could potentially regulate reproductive function at the level of pituitary gonadotrope. Using Cre/Lox technology, we deleted the Kiss1r gene in pituitary gonadotropes (PKiRKO). PKiRKO males have normal genital development (anogenital distance WT: 19.1 ± 0.4 vs. PKiRKO: 18.5 ± 0.4 mm), puberty onset, testes cell structure on gross histology, normal testes size, and fertility. PKiRKO males showed significantly decreased serum FSH levels compared to WT males (5.6 ± 1.9 vs. 10.2 ± 1.8 ng/ml) with comparable LH (1.1 ± 0.2 vs. 1.8 ± 0.4 ng/ml) and testosterone levels (351.8 ± 213.0 vs. 342.2 ± 183.0 ng/dl). PKiRKO females have normal puberty onset, cyclicity, LH and FSH levels and fertility. Overall, these findings indicate that absence of pituitary Kiss1r reduces FSH levels in male mice without affecting testis function. PKiRKO mice have normal reproductive function in both males and females.

Abstract Tipifarnib, a drug that targets HRAS through inhibiting farnesyl transferase, is in phase II clinical trials and appears to show activity in tumors harboring oncogenic HRAS mutations (https://kuraoncology.com/pipeline/#tipifarnib). Unfortunately, these drugs are not expected to be effective on KRAS cancers because KRAS, unlike HRAS, can be prenylated by geranylgeranyl transferase following farnesyl transferase inhibition. To address this, we have developed compounds that prevent farnesylation of KRAS 4B by covalent reaction with C185, the site of prenylation. These compounds bind to a pocket in the G-domain that is formed by interaction with the hypervariable region, an interaction that does not seem to occur in other RAS proteins. This existence of this pocket has been demonstrated through biochemical and biophysical analysis, including NMR and small-angle X-ray scattering. The compounds we have developed are active in cells: they prevent proliferation of MEFs driven by oncogenic KRAS proteins but do not affect MEFs supported by myristoylated KRAS G12D C185S at equivalent concentrations. We are currently optimizing these compounds for further preclinical development. Citation Format: Anna Maciag, Yue Yang, David Turner, Marcin Dyba, Vandana Kumari, Brian Smith, Lixin Fan, Stephan Gysin, Andrew Wolfe, Hazem Abdelkarim, Vadim Gaponenko, Felice Lightstone, Dwight Nissley, Frank McCormick. Preventing KRAS processing [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA21.

Pancreatic ductal adenocarcinomas (PDACs) have enhanced nutrient uptake requirements and rapid metabolic processing. The enzyme UDP-glucose pyrophosphorylase 2 (UGP2) rests at the convergence of multiple metabolic pathways, however the role of UGP2 in tumor maintenance and cancer metabolism remains unclear. Here, we identify an essential role for UGP2 in the maintenance of PDAC growth in both in vitro and in vivo tumor models. Transcription of UGP2 is directly regulated by the YAP/TEAD complex. Loss of UGP2 leads to decreased intracellular glycogen and defects in N-glycosylation targets important for cell growth including epidermal growth factor receptor (EGFR). In murine xenograft models, knockdown of UGP2 halted tumor growth and repressed expression of EGFR. The critical roles of UGP2 in cancer maintenance, metabolism, and protein glycosylation may offer new avenues of therapy for otherwise intractable PDACs.Impact Statement Convergent findings reveal that UDP-glucose pyrophosphorylase 2 has a central role in growth and metabolism of pancreatic ductal adenocarcinomas, highlighting novel therapeutic possibilities for this deadly cancer.### Competing Interest StatementM.S. has received research funds from Puma Biotechnology, AstraZeneca, Daiichi Sankyo, Immunomedics, Targimmune and Menarini Ricerche. He is in the scientific advisory board of Menarini Ricerche and Bioscience Institute and is a cofounder of medendi.org. F.M. is a consultant for Daiichi-Sankyo, Pfizer, Amgen and has received research funding from Daiichi Sankyo. The other authors declare no potential conflicts of interest.

Myxoma virus is a member of Leporipoxviridae whose tropism is tightly restricted to lagomorphs. In susceptible Oryctolagus rabbits, the virus causes a highly lethal disease known as myxomatosis, which begins as a localized infection but rapidly disseminates throughout the animal, leading to immune compromise, mucosal infections, multiorgan failure, and death. In a research setting, myxoma infection of susceptible Oryctolagus cuniculus rabbits is used as a model of poxviral disease progression and represents one of only a few means to study the pathogenesis of this viral family in a native host species. However, the rapid progression of myxomatosis makes accurate prediction of humane endpoints critical to limiting animal pain and distress and preventing death as an endpoint. Here we present case studies of myxomatosis at 2 institutions and offer a refined scoring system to reliably track the course of disease in susceptible rabbits infected with myxoma virus.

The hypothalamic‐pituitary‐gonadal (HPG) axis controls the development and maintenance of reproductive function. The HPG axis is comprised of gonadotropin‐releasing hormone (GnRH) neurons in the hypothalamus that regulate the gonadotrophs in the anterior pituitary gland, stimulating the secretion of luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) into the bloodstream. LH and FSH play essential roles in spermatogenesis in males, folliculogenesis and ovulation in females, and steroidogenesis in both sexes. Recently, kisspeptin (KISS1)/kisspeptin receptor (KISS1R) signaling in GnRH neurons has been shown by our group and others to play an essential role in HPG axis function. However, whether kisspeptin signaling via the Kiss1r affects reproductive function at the level of pituitary is not yet known. Using Cre/Lox technology, we knocked out the Kiss1r gene specifically in pituitary gonadotropes (PKiRKO) by crossing a αGSUCre mouse with a floxed Kiss1r mouse. Q‐RT‐PCR and immunohistochemistry were used to demonstrate a disruption in pituitary Kiss1r mRNA and KISS1R protein in PKiRKO mice relative to controls. Q‐RT‐PCR demonstrated a reduction in Kiss1r mRNA by 88% and 64% in the pituitary of male and female PKiRKO mice (n=8), respectively, compared with wild type (WT) mice (n=8). Immunostaining for KISS1R protein levels exhibited similar trends for protein knock down as observed for the relative mRNA levels. Our results revealed no difference in the age of puberty between WT and PKiRKO littermates, as assessed by the ages of vaginal opening, and first estrus for female mice, and preputial separation for male mice. Furthermore, we saw no difference in ovarian and testes weight respectively in female and male mice. While there were no differences in basal LH and FSH levels, upon performing a GnRH stimulation test in vivo, we observed a significant attenuation ( P <0.05) in stimulated luteinizing hormone (LH) levels in PKiRKO male mice compared with WT male mice, while stimulated LH and FSH levels were no different between WT and PKiRKO female mice. To directly assess the effects of KISS1 on pituitary gonadotroph function that are mediated via the KISS1R we sought to develop an in vitro primary culture system. To test the system, a GnRH dose response and time course study was performed on dispersed and adherent pituitary cells harvested from WT male and female mice. Groups of cells were treated with either 5nM, 30nM or 100nM GnRH, for a duration of 30 and 60 minutes. A significant increase in LH levels was observed in both male and female mice pituitary cells when treated with either 30nM or 100nM GnRH for 60 minutes. This culture system was then applied to calcium flux assays using the calcium indicator dye Fluo‐1. These calcium flux assays indicated that the WT male pituitaries were more responsive to GnRH (100nm) and kisspeptin (1nm) than PKiRKO males. Interestingly a combination of 1nM kp10 and 30nM GnRH potentiated the calcium response in WT males but not their PKiRKO littermates, suggesting that there is interplay of both KISS1 signaling and GnRH signaling at the level of the pituitary to augment pituitary action. These findings indicate overall that the pituitary Kiss1r may plan an important modulatory role and contribute to normal reproductive function. Support or Funding Information National Institutes of Health T32DK007751, JHU UMD Diabetes Research Center (P30 DK079637), RO1 DK101591 and R01HD068777. The American Physiological Society Porter Developmental and Minority Affairs Committee This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

The tumor suppressor PTEN controls cell proliferation by regulating phosphatidylinositol-3-kinase (PI3K) activity, but the participation of PTEN in host defense against bacterial infection is less well understood. Anti-inflammatory PI3K-Akt signaling is suppressed in patients with cystic fibrosis (CF), a disease characterized by hyper-inflammatory responses to airway infection. We found that Ptenl(-/-) mice, which lack the NH2-amino terminal splice variant of PTEN, were unable to eradicate Pseudomonas aeruginosa from the airways and could not generate sufficient anti-inflammatory PI3K activity, similar to what is observed in CF. PTEN and the CF transmembrane conductance regulator (CFTR) interacted directly and this interaction was necessary to position PTEN at the membrane. CF patients under corrector-potentiator therapy, which enhances CFTR transport to the membrane, have increased PTEN amounts. These findings suggest that improved CFTR trafficking could enhance P. aeruginosa clearance from the CF airway by activating PTEN-mediated anti-bacterial responses and might represent a therapeutic strategy.

Studies on the role of hormones in male reproductive aging have traditionally focused on testosterone, but estradiol (E2) also plays important roles in the control of masculine physiology and behavior. Our goal was to examine the effects of E2 on the expression of genes selected for E2-sensitivity, involvement in behavioral neuroendocrine functions, and impairments with aging. Mature adult (MAT, 5 mo) and aged (AG, 18 mo) Sprague-Dawley male rats were castrated, implanted with either vehicle or E2 subcutaneous capsules, and euthanized one month later. Bilateral punches were taken from the bed nucleus of the stria terminalis (BnST), posterodorsal medial amygdala (MePD) and the preoptic area (POA). RNA was extracted, and expression of 48 genes analyzed by qPCR using Taqman low-density arrays. Results showed that effects of age and E2 were age- and region-specific. In the POA, 5 genes were increased with E2 compared to vehicle, and there were no age effects. By contrast the BnST showed primarily age-related changes, with 6 genes decreasing with age. The MePD had 5 genes that were higher in aged than mature males, and 17 genes with significant interactions between age and E2. Gene families identified in the MePD included nuclear hormone receptors, neurotransmitters and neuropeptides and their receptors. Ten serum hormones were assayed in these same males, with results revealing both age- and E2-effects, in several cases quite profound. These results support the idea that the male brain continues to be highly sensitive to estradiol even with aging, but the nature of the response can be substantially different in mature and aging animals.

Abstract We report a mechanism of translational control that is determined by a requirement for eIF4A RNA helicase activity and underlies the anticancer effects of Silvestrol and related compounds. Briefly, activation of cap-dependent translation contributes to T-cell leukemia (T-ALL) development and maintenance. Accordingly, inhibition of translation initiation factor eIF4A with Silvestrol produces powerful therapeutic effects against T-ALL in vivo. We used transcriptome-scale ribosome footprinting on Silvestrol-treated T-ALL cells to identify Silvestrol-sensitive transcripts and the hallmark features of eIF4A-dependent translation. These include a long 5 UTR and a 12-mer sequence motif that encodes a guanine quartet (CGG)4. RNA folding algorithms as well as experimental evidences pinpoint the (CGG)4 motif as a common site of RNA G-quadruplex structures within the 5 UTR. In T-ALL these structures mark approximately eighty highly Silvestrol-sensitive transcripts that include key oncogenes and transcription factors and contribute to the drug's anti-leukemic action. Hence, the eIF4A-dependent translation of G-quadruplex containing transcripts emerges as a gene-specific and therapeutically targetable mechanism of translational control. Citation Format: Kamini Singh, Andrew L. Wolfe, Yi Zhong, Gunnar Rätsch, Hans-Guido Wendel. The 5 UTR of many oncogenes and transcription factors encodes a targetable dependence on the eIF4A RNA helicase. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B23.

The posttranscriptional control of gene expression by microRNAs (miRNAs) is highly redundant, and compensatory effects limit the consequences of the inactivation of individual miRNAs. This implies that only a few miRNAs can function as effective tumor suppressors. It is also the basis of our strategy to define functionally relevant miRNA target genes that are not under redundant control by other miRNAs. We identified a functionally interconnected group of miRNAs that exhibited a reduced abundance in leukemia cells from patients with T cell acute lymphoblastic leukemia (T-ALL). To pinpoint relevant target genes, we applied a machine learning approach to eliminate genes that were subject to redundant miRNA-mediated control and to identify those genes that were exclusively targeted by tumor-suppressive miRNAs. This strategy revealed the convergence of a small group of tumor suppressor miRNAs on the Myb oncogene, as well as their effects on HBP1, which encodes a transcription factor. The expression of both genes was increased in T-ALL patient samples, and each gene promoted the progression of T-ALL in mice. Hence, our systematic analysis of tumor suppressor miRNA action identified a widespread mechanism of oncogene activation in T-ALL.

Polycystic ovary syndrome is the major cause of infertility in reproductive aged women. Polycystic ovary syndrome is associated with high circulating levels of androgens and impaired metabolic function. The goal of this study was to understand how androgen signaling via the androgen receptor (AR) affects reproductive function. We knocked out the AR gene specifically in pituitary gonadotropes (PitARKO) to explore the role of androgen on the development of reproductive function in female mice. There was no difference in the age of puberty between control and PitARKO littermates, which was assessed by the ages of vaginal opening and first estrus. Cyclicity and fertility were also studied, and there was no significant difference between control and PitARKO mice. We observed a significant decrease in basal FSH serum and mRNA levels with no corresponding change in LH serum and mRNA levels. Although the numbers of litters born to control and PitARKO females were the same, the litter size was significantly smaller for PitARKO mice. The LH and FSH responses to ovariectomy was altered with reduced LH/FSH hormone and mRNA levels in PitARKO females. This reduction may be due to reduced expression of activin A/B and gnrhr. The preovulatory surge levels of LH and FSH were dramatically lower in PitARKO mice. The number of corpora lutea was decreased whereas the number of antral follicles was similar between control and PitARKO mice. Overall the pituitary AR contributes to the elaboration of the LH surge and normal reproductive function by regulating LH/FSH expression and secretion.

Abstract The risk of occurrence of breast cancer changes at menopause in women. Various studies propose an association between mammographic density and adipose tissue distribution in premenopausal and post-menopausal women. There is a positive correlation between mammographic density and visceral adiposity in post-menopausal women, but an inverse correlation of the two in premenopausal women. Similar findings have also been discovered in rodent models. To model the premenopausal and post-menopausal status in women, this study used a middle-aged ovariectomized (OVX) Sprague Dawley rat model, given 17b-estradiol (E2) or vehicle (VEH) capsule, E2-deprived rats. Rats were OVX at 11 mo, and given E2 or VEH for 3 or 6 mo. A subset of rats were initially given E2 or VEH for 3 mo, then switched to the opposite treatment for another 3 mo, to evaluate the importance of E2 timing/duration. Mammary tumorigenesis was monitored through the treatment. Tumors were surgically removed upon detection. Rats were euthanized at 14 months old (3 mo of treatment) or 17 months old (6 mo of treatment). The 4th mammary gland was whole mounted for mammarographic density studies and comparisions. Serum estradiol, progesterone and prolactin concentrations were measured. Our results showed: 1) Body weight and visceral fat significantly decreased in E2 compared to VEH treated rats. 2) Following 3 or 6 mo E2 treatment, 3/54 and 5/72 rats, respectively, developed palpable mammary tumors. By contrast, 0/54 and 1/72 rats developed palpable tumors following 3 or 6 mo VEH treatment. In the group given 3 mo VEH then switched to E2 for 3 mo, 2/18 rats developed tumors. 1/18 rats developed tumors in the group given 3 mo of E2 then switched to VEH for 3 mo. 3) Mammary gland whole mounts showed increased mammographic density and decreased fat pad in E2 treated group. 4) Serum prolactin concentration significantly increased in E2 compared to VEH treated rats at euthanization. These results suggest timing and duration of hormone treatments have differing effects on mammary morphology and tumorigenesis. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-10-03.