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Adenoid cystic carcinoma (ACC) is a rare salivary cancer. The highest rates of disease recurrence are in patients with NOTCH pathway activation, reported in up to 20%. Novel drugs targeting NOTCH signaling are under investigation in the recurrent/metastatic (R/M) setting. To understand their clinical utility, there is an urgent need to better characterize the disease course and outcomes following current standard of care treatment.120 patients with R/M ACC underwent clinical review at a single UK Cancer Centre. Patients were retrospectively assessed for tumor NOTCH pathway activation using next generation sequencing (NGS) targeting NOTCH1/2/3 genes and/or NOTCH1 intra-cellular domain (NICD1) immunohistochemistry. Demographic and treatment data were extracted from the clinical notes. Kaplan-Meier survival analysis was performed using log rank test.NOTCH pathway activation was identified in 13/120 patients (11 %). In 12/101 patients analyzed by NGS, NOTCH1/3 activating somatic mutations were identified, and a further patient was identified with NICD1 diffuse nuclear staining in whom NGS testing was not possible. Patients with NOTCH pathway activation had shorter median RFS (1.1 vs 3.4 years, p = 0.2032) and significantly reduced median OS from diagnosis (4.0 vs 16.3 years, p 0.0001). There was significantly reduced median OS from time of disease recurrence/metastasis (1.9 vs 9.6 years, p 0.0001).This study clearly demonstrates a reduction in OS from time of first confirmed disease recurrence/metastasis for patients with NOTCH pathway activated ACC. This provides support for developing new drugs for this sub-group of patients, for whom clinical outcomes are significantly worse and effective treatments are lacking.

6072 Background: ACC is a rare salivary cancer for which effective drug therapies remain lacking. The highest rates of disease recurrence are in patients with NOTCH pathway activation, which is reported in 10-20% of ACC tumors. Novel drugs targeting NOTCH signaling are under investigation in the recurrent and metastatic setting. To understand their clinical utility, there is an urgent need to better characterize the disease course and outcomes following current standard of care treatment from diagnosis and following recurrence. Methods: 120 patients with ACC underwent clinical review at a single UK Cancer Centre from 2017-19. Patients were retrospectively assessed for tumor NOTCH pathway activation using next generation sequencing (NGS) targeting NOTCH1/2/3 genes (n = 98) and/or by immunohistochemistry (IHC) for the NOTCH1 intra-cellular domain (NICD1) (n = 87). To understand the disease course with NOTCH pathway activation, treatment data including surgery, radiotherapy and systemic therapies were extracted and presented as swimmer plots. Kaplan-Meier survival analysis was performed and a difference in survival with/without NOTCH activation was calculated with log rank test. Overall survival (OS) was calculated both from diagnosis and from first confirmed disease recurrence or metastasis, and recurrence free survival (RFS) calculated from diagnosis. Results: Of 120 patients, median age was 46 years (22-74 years). 114/120 patients (95%) had confirmed disease recurrence at clinical review. The primary site was major salivary gland in 58/120 (48%), the others were minor salivary. NOTCH1/3 activating somatic mutations were identified in 11% by NGS (11/98) and NICD1 diffuse nuclear staining was seen in 6% by IHC (5/87) for overall NOTCH activation in 11% (13/120). In NOTCH activated ACC, primary site was major salivary gland in 7/13 (54%), and non-pulmonary visceral/bone metastases were present in 6/13 (46%). Consistent with other reports, patients with NOTCH activation (n = 13) had shorter RFS (0.9 vs 3.6 years, p = 0.11) and significantly reduced OS from diagnosis (4.0 vs 16.3 years, p < 0.0001). Critically, as therapies targeting NOTCH signaling are being evaluated in recurrent/metastatic ACC, there was significantly reduced OS from time of first confirmed disease recurrence or metastasis (1.5 vs 9.6 years, p < 0.0001). This reduction in OS for NOTCH activation following recurrence was seen consistently whether patients were classified using NGS (1.9 vs 9.6 years, p = 0.0009) or NICD1 IHC (0.8 vs 8.5 years, p < 0.0001). Conclusions: This is the first study to report clinical outcomes for patients with NOTCH pathway activated ACC following disease recurrence. Although ACC is frequently considered an indolent disease, the short survival in this sub-group of ACC patients demonstrates the urgent need to develop effective drug therapies in this setting.

Goal: To evaluate Notch inhibitor monotherapy and combination (combo) therapy in Adenoid Cystic Carcinoma (ACC) Patient Derived Xenograft (PDX) models with and without activating Notch mutations (mt). ACC is a rare salivary gland malignancy with no standard of care. Chemotherapy resistance limits treatment to surgery/radiation and ~60% of patients (pts) will recur. ~22% of ACC pts have Notch activating mutations that are associated with a distinct phenotype, aggressive disease and poor prognosis. In a P1 solid tumor study, 2 ACC pts were treated with AL101, an investigational gamma secretase inhibitor (J Clin Oncol 36, 2018 abstract 2515). One pt with an NRR (Negative Regulatory Region) activating mutation had a prolonged partial response, the 2nd pt with a PEST mutation had stable disease. Both had an extended PK profile with a sustained pharmacodynamic response (>50% inhibition of HES1 in peripheral blood). These results prompted us to evaluate the effect of AL101 in tumors that harbor/lack Notch activating mutations. Design: 4 ACC PDX models were evaluated: ACCx9 (Notch1 mt: NRR activating mutation), ACCx11 (Notch1 mt, tandem duplication), ACCx6 (Notch1 wt), ACCx5M1 (Notch1 VUS, not predicted to be activating). Activated Notch1 (nuclear IHC stain) was confined to the Notch mt models. Tumors were implanted into 6-12 week old athymic nude female mice. Upon reaching 150-300 mm3 tumor volume, mice were randomized to treatment (N=5), or vehicle (N=10) arms. AL101 was dosed at 7.5 mg/kg po (days 1-4 of Q7D) as single agent or in combo with cisplatin (3 mg/kg ip; qw) or everolimus (10 mg/kg; po; qd). Results: Significant tumor growth inhibition (TGI) was seen with AL101 monotherapy compared to vehicle treatment in both Notch mt models (ACCx9; 110% TGI P0.99, ACCx5M1: 60% TGI P=0.26). Cisplatin and everolimus monotherapy had no significant effect on Notch mt models (cisplatin: ACCx9; 43% TGI P=0.98, ACCx11; 50% TGI P=0.99, everolimus: ACCx9; 51% TGI P=0.6, ACCx11; 54% TGI P=0.99). AL101 with cisplatin or everolimus had no further benefit in the ACCx9 model (both 106% TGI P=0.98 or P>0.99 respectively). The ACCx11 model had a non-significant benefit (104% TGI P=0.17 for cisplatin combo and 107% TGI P=0.18 for everolimus combo). Interestingly, the ACCx6 Notch wt model was sensitive to everolimus (75% TGI P=0.027) with no significant benefit of adding AL101. Conclusion: AL101 monotherapy had a significant anti-tumor effect in ACC PDX tumors with Notch activating mutations and lacked effectiveness in tumors lacking such mutations. Neither cisplatin nor everolimus monotherapy treatment had a significant effect in Notch mt models, alone or in combination with AL101. These data support the clinical development of AL101 as a targeted monotherapy for ACCs with Notch activating mutations. Citation Format: Renata ferrarotto*, Genia Alpert*, Udi Gluschnaider, Rami Rauch, Adi Mondshine, Oz Solomon, Bill Kramer, Evgeny Izumchenko, John Heymach, Andrea Vergara-Silva, Jon Aster, Matti Davis. AL101 mediated tumor inhibition in Notch mutated ACC PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4885.

1064 Background: The Notch pathway is activated during mammary gland development and has been implicated as a key driver in breast cancer. There is an urgent need to identify new therapeutic strategies for triple-negative breast cancer (TNBC), a sub-type associated with poor prognosis and no available targeted therapies. Notch gain of function (GOF) genetic alterations are potential tumor drivers found in ~10% of TNBC. This motivated the development of Notch inhibitors, including AL101 a pan-Notch, gamma secretase inhibitor (J Clin Oncol 36, 2018 abstract 2515). AL101 is currently being evaluated in Adenoid Cystic Carcinoma patients with activating Notch mutations (NCT03691207, ACCURACY trial). Here, we aim to test the activity of AL101 in TNBC patient derived xenograft (PDX) models with Notch activating genetic alterations. Methods: Gene expression cluster analysis was performed for 38 TNBC PDX tumors using a list of 21 Notch target genes. Seven tumors, bearing a “Notch-on” signature, were enriched with mutated/fusion (M/F) Notch genes and clustered separately from all other tumors. Of 9 models selected for study, 4 had a Notch-on signature and were expected to respond to AL101. Tumors were implanted into female athymic nude mice. Once tumors reached an average size of 150-300 mm3, mice (n = 5/group) were randomized to Vehicle or AL101 treatment arms (3 mg/kg, PO, 4on/3off) until tumors reached 1500 mm3 or day 60. Results: As measured by tumor growth inhibition (TGI), AL101 was more potent in tumors with a putative Notch-on signature. Within these 4 models, M/F genes were present in Notch1-NRR GOF (103% TGI p = 0.0004); Notch2-fusion (62%TGI p = 0.036); Notch3-fusion (75% TGI p = 0.032); or Notch4-fusion (147% TGI p < 0.00001). Tumors lacking the Notch signature did not respond significantly to AL101: WT Notch (43% TGI p = 0.0104; 64% TGI p = 0.13); Notch1 with a predicted loss of function mutation (12% TGI p = 0.53), Notch1 Variant of Unknown Significance (VUS) (30% TGI p = 0.44), Notch2 VUS (41% TGI p = 0.44). Conclusions: We demonstrate that in TNBC PDX models, the presence of a Notch-on signature and Notch GOF mutations/fusions correlates with potent response to AL101. These data support the clinical development of AL101 as a targeted therapy for TNBC with Notch GOF alterations.

Mammalian oocytes remain dormant in the diplotene stage of prophase I until the resumption of meiosis characterized by germinal vesicle breakdown (GVBD) following the preovulatory gonadotropin stimulation. Based on genome-wide analysis of peri-ovulatory DNA microarray to identify paracrine hormone-receptor pairs, we found increases in ovarian transcripts for endothelin-1 and endothelin receptor type A (EDNRA) in response to the preovulatory luteinizing hormone (LH)/human chorionic gonadotropin (hCG) stimulation. Immunohistochemical analyses demonstrated localization of EDNRA in granulosa and cumulus cells. In cultured preovulatory follicles, treatment with endothelin-1 promoted oocyte GVBD. The stimulatory effect of endothelin-1 was blocked by cotreatment with antagonists for the type A, but not related type B, receptor. The stimulatory effect of hCG on GVBD was partially blocked by the same antagonist. The endothelin-1 promotion of GVBD was found to be mediated by the MAPK/ERK pathway but not by the inhibitory G protein. Studies using cumulus–oocyte complexes and denuded oocytes demonstrated that the endothelin-1 actions are mediated by cumulus cells. Furthermore, intrabursal administration with endothelin-1 induced oocyte GVBD in preovulatory follicles. Our findings demonstrate a paracrine role of endothelin-1 in the induction of the resumption of meiosis and provide further understanding on the molecular mechanisms underlying the nuclear maturation of oocytes induced by the preovulatory LH surge.

Optimal maturation of oocytes and successful development of preimplantation embryos is essential for reproduction. We performed DNA microarray analyses of ovarian transcripts and identified glial cell line-derived neurotrophic factor (GDNF) secreted by cumulus, granulosa, and theca cells as an ovarian factor stimulated by the preovulatory LH/hCG surge. Treatment of cumulus–oocyte complexes with GDNF enhanced first polar body extrusion with increase in cyclin B1 synthesis and the GDNF actions are likely mediated by its receptor GDNF family receptor-alpha1 (GFRA1) and a co-receptor ret proto-oncogene (Ret), both expressed in oocytes. However, treatment with GDNF did not affect germinal vesicle breakdown and cytoplasmic maturation of oocytes. During the preimplantation stages, GDNF was expressed in pregnant oviducts and uteri, whereas GFRA1 and Ret were expressed in embryos throughout early development with an increase after the early blastocyst stage. In blastocysts, both GDNF and GFRA1 were exclusively localized in trophectoderm cells, whereas Ret was detected in both cell lineages. Treatment with GDNF promoted the development of two-cell-stage embryos into blastocysts showing increased cell proliferation and decreased apoptosis mainly in trophectoderm cells. Our findings suggest potential paracrine roles of GDNF in the promotion of completion of meiosis I and the development of early embryos.

Sequencing of genomes from diverse organisms facilitates studies on the repertoire of genes involved in intercellular signaling. Extending previous efforts to annotate most human plasma membrane receptors in the Human Plasma Membrane Receptome database, we matched cognate ligands with individual receptors by surveying the published literature. In the updated online database we called “liganded receptome,” users can search for individual ligands or receptors to reveal their pairing partners and browse through receptor or ligand families to identify relationships between ligands and receptors in their respective families. Because local signaling systems are prevalent in diverse normal and diseased tissues, we used the liganded receptome knowledgebase to interrogate DNA microarray datasets for genome-wide analyses of potential paracrine/autocrine signaling systems. In addition to viewing ligand-receptor coexpression based on precomputed DNA microarray data, users can submit their own microarray data to perform online genome-wide searches for putative paracrine/autocrine signaling systems. Investigation of transcriptome data based on liganded receptome allows the discovery of paracrine/autocrine signaling for known ligand-receptor pairs in previously uncharacterized tissues or developmental stages. The present annotation of ligand-receptor pairs also identifies orphan receptors and ligands without known interacting partners in select families. Because hormonal ligands within the same family usually interact with paralogous receptors, this genomic approach could also facilitate matching of orphan receptors and ligands. The liganded receptome is accessible at http://receptome.stanford.edu.

Ovarian Kaleidoscope database (OKdb) is an online, searchable, public database containing text-based and DNA microarray data to facilitate research by ovarian researchers. Using key words and predetermined categories, users can search ovarian gene information based on gene function, cell type of expression, cellular localization, hormonal regulation, mutant phenotypes, chromosomal location, ligand-receptor relationship, and other criteria, either alone or in combination. For individual genes, users can access more than 10 extensive DNA microarray datasets to interrogate gene expression patterns in a development-specific and cell type-specific manner. All ligand and receptor genes expressed in the ovary are matched to facilitate investigation of paracrine/autocrine signaling. More than 3500 ovarian genes in the database are matched to 185 gene pathways in the Kyoto Encyclopedia of Genes and Genomes to allow for elucidation of gene interactions and relationships. In addition to >400 genes with infertility or subfertility phenotypes when mutated in mice or humans, the OKdb also lists ∼50 and ∼40 genes associated with polycystic ovarian syndrome and primary ovarian insufficiency, respectively. The expanding OKdb is updated weekly and allows submission of new genes by ovarian researchers to allow instant access to DNA microarray datasets for newly submitted genes. The present database is a virtual community for ovarian researchers and allows users to instantaneously provide their comments for individual gene pages based on an automated Web-discussion system. In the coming years, we will continue to add new features to serve the ovarian research community.

The mission of the Encyclopedia of DNA Elements (ENCODE) Project is to enable the scientific and medical communities to interpret the human genome sequence and apply it to understand human biology and improve health. The ENCODE Consortium is integrating multiple technologies and approaches in a collective effort to discover and define the functional elements encoded in the human genome, including genes, transcripts, and transcriptional regulatory regions, together with their attendant chromatin states and DNA methylation patterns. In the process, standards to ensure high-quality data have been implemented, and novel algorithms have been developed to facilitate analysis. Data and derived results are made available through a freely accessible database. Here we provide an overview of the project and the resources it is generating and illustrate the application of ENCODE data to interpret the human genome., National Human Genome Research Institute (U.S.), National Institutes of Health (U.S.)

Development of early embryos is regulated by autocrine/paracrine factors. Analyzing the expression of polypeptide ligand–receptor pairs using DNA microarray datasets, we identified transcripts for artemin, a member of the GDNF (glial cell line-derived neurotrophic factor) family, its receptor GFRA3 (GDNF family receptor-alpha 3) and coreceptor RET. Here we report an autocrine/paracrine role of the artemin-GFRA3 signaling system in regulating early embryonic development and apoptosis. Possible involvement of the MAP kinase signaling pathway was also demonstrated. The genome-wide survey of ligand–receptor pairs and early embryo cultures provided a better understanding of autocrine/paracrine embryonic factors important for optimal blastocyst development.

Background Shortly after stimulation by the preovulatory surge of luteinizing hormone (LH), oocytes arrested at the late prophase I resume meiosis characterized by germinal vesicle breakdown (GVBD), chromosome condensation, and extrusion of the first polar body in preparation for fertilization and early embryonic development. However, oocytes express few or no LH receptors and are insensitive to direct LH stimulation. Thus, factors released by granulosa or theca cells expect to convey the LH stimuli to oocytes. To identify candidate ligand-receptor pairs potentially involved in the process of oocyte maturation, we performed DNA microarray analyses of ovarian transcripts in mice and identified Kit ligand (Kitl) as an ovarian factor stimulated by the LH/hCG surge. The purpose of this study is to investigate the roles of KITL in the nuclear and cytoplasmic maturation of preovulatory mouse oocytes. Methods The levels of Kitl and c-kit transcripts in mouse ovaries and isolated ovarian cells were determined by real-time RT-PCR, while expression of KITL protein was examined by immunohistochemistry. Follicle culture, cumulus-oocyte complexes (COC) and denuded oocytes culture were used to evaluate the effect of KITL on mouse oocyte nuclear maturation. To assess the effect of KITL treatment on the cytoplasmic maturation of preovulatory oocytes, we performed in vitro maturation of oocytes followed by in vitro fertilization. Results Major increase of Kitl transcripts in granulosa cells and mouse ovaries, and predominant expression of c-kit in preovulatory oocytes were identified by real-time RT-PCR. Predominant expression of KITL protein was found in granulosa cells of preovulatory and small antral follicles at 4 h after hCG treatment. In vitro cultures demonstrated that treatment with KITL enhanced first polar body extrusion in a dose-dependent manner. Moreover, treatment of COC with KITL enhanced first polar body extrusion with increase in cyclin B1 synthesis which is important for the progression of meiotic maturation after GVBD. In contrast, treatment of cultured preovulatory follicles with KITL did not affect GVBD and KITL has no effect on cytoplasmic maturation of preovulatory oocytes. Conclusion Our findings suggest potential paracrine roles of KITL in the nuclear maturation of preovulatory oocytes by promoting first polar body extrusion.

In addition to gonadotropins, many ovarian paracrine factors are crucial for optimal follicle rupture, oocyte maturation, and luteinization. Based on DNA microarray analyses, we found that transcripts for the fibroblast growth factor-inducible-14 (Fn14) receptor are increased after LH/human chorionic gonadotropin (hCG) treatment of gonadotropin-primed immature mice or rats. Fn14 is the cognate receptor for TNF-related weak inducer of apoptosis (TWEAK), a TNF superfamily member. TWEAK transcripts also were detected in the ovary; however, their levels were not regulated by gonadotropins. In situ hybridization analyses indicated that the Fn14 receptor is expressed in the granulosa and cumulus cells of preovulatory follicles and, to a lesser extent, in theca cells. In contrast, in situ hybridization analyses revealed that TWEAK is primarily expressed in theca cells. In cultured granulosa cells pretreated with hCG to induce Fn14 receptor expression, treatment with TWEAK suppressed progesterone synthesis without accompanying changes in cAMP production. Furthermore, intrabursal injection of TWEAK suppressed ovarian progesterone content in gonadotropin-primed rats. In contrast, preovulatory follicles cultured in the presence of the Fn14 decoy, a recombinant protein containing the ligand-binding domain of Fn14, led to increases in progesterone production, presumably by antagonizing the actions of endogenous TWEAK. Likewise, ip injection of the Fn14 decoy enhanced serum progesterone levels with accompanying increases in transcript levels for several key steroidogenic enzymes. The present findings demonstrate a suppressive role of the TWEAK/Fn14 signaling system in the ovary. Following gonadotropin induction of ovulation, Fn14 is induced and could protect preovulatory follicles from excessive luteinization.

Ghrelin, a circulating appetite-inducing hormone, is derived from a prohormone by posttranslational processing. On the basis of the bioinformatic prediction that another peptide also derived from proghrelin exists, we isolated a hormone from rat stomach and named it obestatin—a contraction of obese, from the Latin “obedere,” meaning to devour, and “statin,” denoting suppression. Contrary to the appetite-stimulating effects of ghrelin, treatment of rats with obestatin suppressed food intake, inhibited jejunal contraction, and decreased body-weight gain. Obestatin bound to the orphan G protein–coupled receptor GPR39. Thus, two peptide hormones with opposing action in weight regulation are derived from the same ghrelin gene. After differential modification, these hormones activate distinct receptors.

Intercellular communication in multicellular organisms requires the relay of extracellular signals by cell surface proteins to the interiors of cells. The availability of genome sequences from humans and several model organisms has facilitated the identification of several human plasma membrane receptor families and allowed the analysis of their phylogeny. This review provides a global categorization of most known signal transduction-associated receptors as enzymes, recruiters, and latent transcription factors. The evolution of known families of human plasma membrane signaling receptors was traced in current literature and validated by sequence relatedness. This global analysis reveals themes that recur during receptor evolution and allows the formulation of hypotheses for the origins of receptors. The human receptor families involved in signaling (with the exception of channels) are presented in the Human Plasma Membrane Receptome database .