Ulipristal acetate decreases active TGF-β3 and its canonical signaling in uterine leiomyoma via two novel mechanisms

for 12 weeks (one treatment course [TC] for VENUS I and two TCs for VENUS II, separated by a drug-free interval of two menses), with a 12week drug-free follow-up. Patients with abnormal liver chemistries at screening were excluded, ie, R2  upper limit of normal (ULN) for alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), or total bilirubin. MATERIALS AND METHODS: Liver tests were conducted at screening, baseline, end of TC1 (VENUS I and II), 10-18 days after first menses following TC2 (VENUS II), and end of study (or early withdrawal), and were assessed in the safety population, comprising all randomized patients who received R1 UPA dose. Cases of Hy’s Law were defined as post-baseline elevation of ALT or AST R3  ULN, with bilirubin R2  ULN and ALP <2  ULN. Liver chemistry discontinuation rules were: 1) ALT R3  ULN and bilirubin R2  ULN; 2) ALT R5  ULN; 3) ALT R3  ULN plus symptoms; 4) ALT R3  ULN for R4 weeks; 5) ALT R3  ULN without weekly monitoring for 4 weeks. To further assess UPA effects on the liver, international criteria for drug-induced liver injury were applied, defined as any one of the following: 1) ALT R5  ULN; 2) ALP R2  ULN; or 3) ALT R3  ULN and total bilirubin >2  ULN [1]. RESULTS: The analysis population comprised 554 patients in TC1 and 313 in TC2. Liver test data following TC1 and following TC2 are presented in Table 1. No patients met liver chemistry study discontinuation criteria or Hy’s Law criteria. No patients had ALT R5  ULN or ALT R3  ULN and total bilirubin >2  ULN. One patient, in the UPA 10 mg arm of VENUS I, had ALP R2  ULN at the end of treatment; however, this abnormality was present at baseline. One patient receiving pbo also had ALP R2  ULN at the end of treatment in VENUS II. CONCLUSIONS: In VENUS I and II, there was no evidence for UPAinduced liver injury. References: 1. Aithal GP, Watkins PB, Andrade RJ, et al. Case definition and phenotype standardization in drug-induced liver injury. Clin Pharmacol Ther 2011;89:806-15. Supported by: Allergan plc, Dublin, Ireland. Editorial assistance: Complete HealthVizion. P-66 Tuesday, October 9, 2018 6:30 AM ULIPRISTAL ACETATE DECREASES ACTIVE TGF-b3 AND ITS SIGNALING THROUGH UP-REGULATION OF FIBRILLIN. T. D. Lewis,a M. Malik,b J. Britten-Webb,c J. Pilgrim,d T. Parikh,e J. M. Aly,f W. Catherino.g aProgram in Reproductive Endocrinology & Gynecology (PREG), NIH, Upper Marlboro, MD; bUSUHS, Bethesda, MD; cObstetrics and Gynecology, Uniformed Services University of the H, Bethesda, MD; dNational Institutes of Health, Bethesda, MD; eNational Institutes of Health, Silver Spring, MD; fUSUHS, NIH, Bethesda, MD; gUniformed Services University of the Health Sciences, Bethesda, MD. OBJECTIVE: Fibrosis inducing pathways, namely Transforming Growth Factor (TGF) signaling, has been implicated in the induction and maintenance of leiomyoma. Previous work has shown that treatment with the Selective Progesterone Receptor Modulator ulipristal acetate (UPA) decreases components of the extracellular matrix (ECM) thereby reducing leiomyoma bulk. Herein, we evaluate the effects of UPA on TGF family member expression and on TGF signaling pathways. DESIGN: Molecular analysis of human tissue and a 2D immortalized cell culture. MATERIALS AND METHODS: RNASeq was performed on matched myometrium & leiomyoma of patients treated with placebo or UPA. Quantitative real-time Polymerase Chain Reaction (qPCR) was utilized to assess fold gene differences in expression of genes involved in TGF signaling. Western immunoblot analysis was then performed to evaluate expression of TGF-b1 & b3; Total & Phosphorylated TGF Receptor II (TGFRII), SMAD 2/3, AKT, and ERK 1/2 in tissue samples. Components of the ECM (including collagen 1A, fibronectin, fibromodulin, versican, & fibrillin) were also evaluated. Attention then turned to a 2D immortalized leiomyoma cell line model treated with UPA 10-10 M through 10-6 M. qPCR and Western immunoblotting were employed to assess fold gene and protein expression of the aforementioned targets. RESULTS: RNASeq analysis of tissue samples revealed a statistically significant increase in TGFb1 & b3 mRNA transcripts in leiomyoma, as compared against patient matched myometrium. However, an unpaired analysis comparing placebo to UPA treated leiomyoma showed no statistical difference in mRNA transcripts following three months of UPA treatment. FERTILITY & STERILITYÒ Western immunoblot analysis of protein extracted from UPA treated samples revealed significant reductions of active TGF-b3, phosphorylated TGFRII and phosphorylated SMAD 2/3. UPA treatment lead to statistically significant reductions of collagen 1A, fibronectin, fibromodulin, & versican. Interestingly, there was a statistically significant increase in fibrillin expression in leiomyoma treated with UPA. Data from in-vitro assays were consistent with findings from our in-vivo studies. CONCLUSIONS: Members of the TGF family are secreted as inactive complexes bound to Latent TGF Binding Protein. These inactive complexes are secreted into the ECM, where the glycoprotein fibrillin maintains TGF in an inactive conformational state. The current study reveals a significant upregulation of fibrillin protein expression in response to UPA providing a novel mechanism whereby UPA leads to decreased active TGF-b3, which therefore leads to diminished canonical pathway signaling. References: Cox J, Malik M, Britten J, Lewis T, Catherino WH. Ulipristal Acetate and Extracellular Matrix Production in Human Leiomyomas in-vivo: A Laboratory Analysis of a Randomized Placebo Controlled Trial. Reprod Sci. 2018. 25(2): 198-206. Lewis TD, Malik M, Britten J, San Pablo AM, Catherino WH. A Comprehensive Review of the Pharmacologic Management of Uterine Leiomyoma. BioMed Research International. 2018. (2018): 1-12. Supported by: Supported by the Intramural Research Program of the Eunice Kennedy Shriver, National Institute of Child Health and Human Development (NICHD). P-67 Tuesday, October 9, 2018 6:30 AM ULIPRISTAL TREATMENT INCREASES FOSB EXPRESSION IN HUMAN LEIOMYOMA SURGICAL SPECIMENS AND CELLS. J. Pilgrim,a J. Arismendi,b T. D. Lewis,c M. Malik,b J. Britten-Webb,d T. Parikh,e J. M. Aly,f W. Catherino.b aNIH, Bethesda, MD; bUSUHS, Bethesda, MD; c NIH, Upper Marlboro, MD; dObstetrics and Gynecology, USUHS, Bethesda, MD; eNIH, Silver Spring, MD; fUSUHS, NIH, Bethesda, MD. OBJECTIVE: To determine whether FosB, a member of the activator protein complex-1 (AP-1), is altered in leiomyoma compared with myometrium, and if expression of FosB is altered with Ulipristal Acetate (UPA) treatment. DESIGN: Molecular analysis of human surgical specimen and two-dimensional immortalized cell culture. MATERIALS AND METHODS: RNA sequence analysis was performed on matched myometrium & leiomyoma of patients treated with placebo or UPA. Quantitative real-time reverse-transcriptase Polymerase Chain Reaction (qrt-PCR) was utilized to assess fold gene differences in expression of the AP-1 family member FosB. Western immunoblot analysis was then performed to evaluate expression of these family members in human tissue. An in-vitro 2D-immortalized leiomyoma cell line model was subsequently treated with UPA at various concentrations. qrt-PCR and Western immunoblotting were utilized to measure fold gene and protein expression of FosB. RESULTS: RNAseq demonstrated a dramatic reduction of FosB transcripts when placebo treated leiomyoma were compared against placebo treated myometrium (0.09 fold; p¼0.003). Conversely, when comparing UPA treated to placebo treated leiomyoma, there was a statistically significant 5.11 fold increase in FosB mRNA transcripts (p¼0.03). Western Immunoblot was used to confirm FosB expression in leiomyoma compared against myometrium; and placebo vs. UPA treated leiomyoma. Surprisingly, in our in-vitro 2D-immortalized leiomyoma cell line model, we noted a 2.0 fold increase in protein expression of FosB when comparing leiomyoma to myometrium. Cells were subsequently treated with UPA at various concentrations and FosB message was affected in a dose responsive manner. CONCLUSIONS: Members of the AP-1 family are nuclear transcription factors that have been associated with cellular proliferation, differentiation and avoidance of cellular death depending on the cell type and dimerization (homo vs hetero) status of the receptor(s). Herein, we report a significant increase in FosB mRNA following treatment with UPA in an in-vivo model. Our in-vitro model revealed a 2.0 fold increase in FosB expression when leiomyoma cells were compared against myometrial cells. Interestingly, UPA treatment revealed dose dependent increases in FosB protein in leiomyoma cells, suggesting possible decelerated destruction of the protein with UPA treatment. Supported by: Supported by the Intramural Research Program of the Eunice Kennedy Shriver, National Institute of Child Health and Human Development (NICHD). e137