Asumda, Faizal Z.

Definitive endoderm (DE) differentiation leads to the development of the major internal organs including the liver, intestines, pancreas, gall bladder, prostate, bladder, thyroid, and lungs. The two primary methods utilized for in vitro differentiation of induced pluripotent stem cells (iPSCs) into DE cells are the growth factor (GF) and the small molecule (SM) approaches. The GSK-3 inhibitor (CHIR99021) is a key factor for the SM approach. Activin A and Wnt3a are utilized in the GF approach. In this study, both the GF and SM protocols were compared to each other. The results show that both the GF and SM protocol produce DE with a similar morphological phenotype, gene and protein expression, and a similar level of homogeneity and functionality. However, on both the gene expression and proteomic level, there is a divergence between the two protocols during hepatic specification. Proteomic analysis shows that hepatoblasts from the GF protocol have significantly differentially expressed proteins (DEPs) involved in liver metabolic pathways compared to the SM protocol. Well-validated DE differentiation protocols are needed to fully unlock the clinical potential of iPSCs. In the first step of generating DE-derived tissue, either protocol can be utilized. However, for hepatic specification, the GF protocol is more effective.

Upamostat is an orally available small-molecule serine protease inhibitor that is a highly potent inhibitor of trypsin 1, trypsin 2, trypsin 3 (PRSS1/2/3), and the urokinase-type plasminogen activator (uPA). These enzymes are expressed in many cancers, especially during tissue remodeling and subsequent tumor cell invasion. Opaganib (ABC294640), a novel, orally available small molecule is a selective inhibitor of the phosphorylation of sphingosine to sphingosine-1-phosphate (S-1-P) by sphingosine kinase 2 (SPHK2). Both sphingosine kinase 1 (SPHK1) and SPHK2 are known to regulate the proliferation-inducing compound S-1-P. However, SPHK2 is more critical in cancer pathogenesis. The goal of this project was to investigate the potential antitumor effects of upamostat and opaganib, individually and in combination, on cholangiocarcinoma (CCA) xenografts in nude mice. PAX165, a patient-derived xenograft (PDX) from a surgically resected CCA, expresses substantial levels of SPHK2, PRSS1, PRSS2, and PRSS3. Four groups of 18 mice each were treated with upamostat, opaganib, both, or vehicle. Mouse weights and PAX165 tumor volumes were measured. Tumor volumes in the upamostat, opaganib, and upamostat plus opaganib groups were significantly decreased compared to the control group.

The PAX1 gene plays an important role in the development of the parathyroid glands and the thymus. Mouse knockout models of PAX1, PAX3, and PAX9 have been found to have hypoplastic or absent parathyroid glands. To our knowledge, there are no reported cases of PAX1-associated hypoparathyroidism in humans. We present a case of hypoparathyroidism in a 23-month-old boy with a homozygous pathogenic variant in the PAX1 gene ( PAX1 NM_006192.5 c.463_465del variant), predicted to cause an in-frame deletion of asparagine at position 155 (p.Asn155del) of the PAX1 protein. The hypoparathyroidism was unmasked after the patient developed significant hypocalcemia while receiving GoLYTELY (polyethylene glycol 3350, sodium sulfate anhydrous, sodium bicarbonate, sodium chloride, potassium chloride) for bowel cleanout. The patient had mild and asymptomatic hypocalcemia prior to hospitalization. The patient was noted to have inappropriately normal parathyroid hormone (PTH) level at the time of documented hypocalcemia thereby suggesting a diagnosis of hypoparathyroidism. Plain language summary The first human case of hypoparathyroidism associated with a rare genetic disorder: a case report of PAX1 gene mutation The paired box ( PAX) gene family is important for embryo development. One subfamily, PAX1, is necessary for development of the spinal column, thymus (important for the immune system development), and parathyroid (helps regulate the amount of calcium in the body). We present the case of a 23-month-old boy with known PAX1 gene mutation who came in with episodes of vomiting and poor growth. His presentation was thought to be most likely related to constipation. He was started on bowel cleanout medication and intravenous fluids. However, his calcium that had been mildly low subsequently dropped to very low levels. The level of parathyroid hormone (which helps regulate calcium levels) was inappropriately normal, meaning that his body was unable to make more, and was consistent with hypoparathyroidism. He was treated with calcium supplements and vitamin D and calcium levels normalized. He continues to be on calcium and vitamin D and calcium levels have remained stable. Doctors should keep this complication in mind when treating patients with PAX1 gene mutation.

Mucopolysaccharidoses (MPS) are a group of inherited lysosomal storage disorders caused by deficient levels and/or activity of glycosaminoglycan (GAG)-degradative enzymes. MPS are characterized by accumulation of the mucopolysaccharides heparan sulfate, dermatan sulfate, keratan sulfate, or chondroitin sulfate in tissues. We report the case of a 38-year-old woman with a history of joint restriction and retinitis pigmentosa who developed bivalvular heart failure requiring surgery. It was not until pathological examination of surgically excised valvular tissue that a diagnosis of MPS I was made. Her musculoskeletal and ophthalmologic symptoms, when placed in the context of MPS I, painted the diagnostic picture of a genetic syndrome that was overlooked until a diagnosis was made in late middle age.

Transforming growth factor-β (TGF-β) activates hepatic stellate cells (HSCs), which drive liver fibrosis via the production and deposition of extracellular matrix (ECM). We aimed to elucidate the mechanistic role of sulfatase-2 (SULF2) in liver fibrosis. To this end, we induced liver fibrosis in wild-type (WT) and SULF2 knockout (Sulf2-KO) mice (6–8 weeks-old) via bile duct ligation (BDL), intraperitoneal injection of carbon tetrachloride (CCl4) or thioacetamide (TAA). The levels of fibrosis in the liver sections were assessed via Sirius red and Masson’s trichrome staining, immunohistochemistry and immunoblotting for α-smooth muscle actin (α-SMA) and hydroxyproline. To evaluate the interaction between TGF-β and SULF2, we transfected human HSCs with scrambled control shRNA and shRNA constructs targeting SULF2 and measured α-SMA expression following treatment with TGF-β1 ligand. We show here that knockout of SULF2 significantly decreases collagen content, as well as bands of bridging fibrosis, as demonstrated by Sirius red, Masson’s trichrome and α-SMA staining after BDL, CCl4 and TAA injection in Sulf2-KO versus WT mice. In all three models of liver fibrosis, we observed significantly lower levels of hydroxyproline in the Sulf2-KO mice compared to the WT mice. HSCs with reduced levels of SULF2 failed to significantly express α-SMA and collagen type I following treatment with TGF-β1. Furthermore, SULF2 co-localizes with TGFBR3 and the in vitro knockdown of SULF2 in HSCs decreases the release of TGF-β1 from TGFBR3. Together, these data suggest that SULF2 regulates liver fibrosis via the TGF-β signaling pathway. Pharmacologic inhibition of SULF2 may represent a novel therapeutic approach to improve liver fibrosis.

Abstract Background: Upamostat is an orally available small molecule serine protease inhibitor that is a highly potent inhibitor of trypsin 1, trypsin 2 and trypsin 3 (PRSS1/2/3) as well as urokinase-type plasminogen activator (uPA) which are expressed in many cancers and mediate cell migration, invasion and tissue remodeling. Opaganib (ABC294640), a novel, orally available small molecule is a specific inhibitor of sphingosine kinase 2 (SPHK2), which phosphorylates sphingosine to sphingosine-1-phosphate (S-1-P). While proliferation induced by S-1-P is regulated by both sphingosine kinase 1 (SPHK1) and SPHK2, SPHK2 appears to be more involved in cancer. We aimed to investigate the potential antitumor effect of upamostat and opaganib, individually and in combination, on cholangiocarcinoma (CCA) patient derived xenografts (PDX) in nude mice. Methods: PAX165, a PDX from a surgically resected CCA, expresses substantial levels of SPHK2, PRSS1, PRSS2 and PRSS3. 4 groups of 18 mice were treated with either drug or both. Mouse weights and tumor volumes were measured. In addition, experiments were conducted using the chorioallantoic membrane (CAM) of chicken embryos. Results: Table 1 shows the average tumor size for the control, upamostat, opaganib, and upamostat+opaganib groups at the study end point (Day 42). Tumor volumes in the upamostat, opaganib, and upamostat+opagnib groups were significantly decreased compared to the control group. The CAM experiments are ongoing and will be presented at the AACR Annual Meeting. Change in tumor volumes (mean) of CCA PDX after opaganib, upamostat or combination treatmentControlOpaganibUpamostatOpaganib+UpamostatPre-treatment129.9128.7118.8126.8Day 42198.6102.093.3186.09Percent change Day 0-42+53%-21%-21%-32%P value vs. control0.00020.00100.0008 Conclusion: This preclinical study demonstrated that upamostat and opaganib resulted in tumor regression in mice. Body weights of the mice showed no significant inter- or intra- group differences. The combination of upamostat and opaganib treatment showed greater regression compared to either upamostat or opaganib alone. Studies are underway to identify the molecular mechanisms of their interaction. Citation Format: Faizal Z. Asumda, Mohamed A. Hassan, Yo Han Kim, Nellie A. Campbell, Xin Luo, Daniel R. O'Brien, Sarah A. Buhrow, Joel M. Reid, Michael J. Moore, Vered Katz Ben-Yair, Reza Fathi, Mark L. Levitt, Fabrice Lucien-Matteoni, Jennifer L. Leiting, Mark J. Truty, Lewis R. Roberts. Effects of upamostat and opaganib on cholangiocarcinoma patient derived xenografts [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3078.

We report for the first time that in wild-type (WT) mice, fast-food diet (FFD) induced a threefold increase in hepatic Sulf2 mRNA and a 2.2-fold increase in sulfatase 2 (SULF2) protein expression compared with WT mice on standard chow diet (SC). We showed that knockout of SULF2 ameliorates FFD-induced obesity, hyperlipidemia, steatohepatitis, and fibrosis. These data, along with work from other laboratories, suggest that SULF2 may be critical to the ability of the liver to progress to nonalcoholic steatohepatitis and fibrosis in conditions of overnutrition.