Combined Antitumor Effect of the Serine Protease Urokinase Inhibitor Upamostat and the Sphingosine Kinase 2 Inhibitor Opaganib on Cholangiocarcinoma Patient-Derived Xenografts

Abstract

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.

Keywords: WX-UK1; cholangiocellular carcinoma; opaganib; patient-derived xenograft (PDX); serine protease; sphingosine kinase; upamostat.

Figure 1

19 RNA sequencing was performed on 19 PDX lines established at the Mayo Clinic. Expressions of sphingosine kinase 2 (SPHK2), trypsin 1 (PRSS1), trypsin 2 (PRSS2), and trypsin 3 (PRSS3) were compared. All 19 PDXs showed expression of SPHK2 and trypsin 3; however, PAX165 showed substantial expression of all 4 proteins, making it ideal for the study.

Figure 2

(A). Representative tumor nodules at the study endpoint are shown. (B). Body weights of the mice in the different groups were monitored, and no significant changes were seen within or between the groups. (C). At the study endpoint, tumor volumes of the upamostat, opaganib, and upamostat plus opaganib treated groups were significantly decreased compared to the control group. p < 0.0001 by t-test. The suppressive effect of upamostat plus opaganib on tumor growth was greater than that of upamostat alone, p < 0.02 by t-test. (D). Tumor volume changes from baseline were monitored. Compared to the control group, upamostat and opaganib each significantly reduced tumor growth in mice (p < 0.0001). When administered together, upamostat plus opaganib suppressed tumor growth to a greater degree than upamostat alone (p = 0.0002).

Figure 3

IHC staining of bile ducts shows decreases in drug target-expressing cells in CCA after treatment. Target: (A). Trypsin 1 (PRSS1), trypsin 3 (PRSS3), and putative trypsin 6 (PRSS3P2); (B). Sphingosine kinase 2 (SPHK2).

Figure 3

IHC staining of bile ducts shows decreases in drug target-expressing cells in CCA after treatment. Target: (A). Trypsin 1 (PRSS1), trypsin 3 (PRSS3), and putative trypsin 6 (PRSS3P2); (B). Sphingosine kinase 2 (SPHK2).

Figure 4

Upamostat, opaganib, and upamostat plus opaganib treatment groups showed significantly less Ki-67 staining, indicating reduced proliferation after treatment, p < 0.0001.

Figure 5

Significantly increased cellular apoptosis after opaganib treatment compared to all other treatment groups.

Figure 6

(A) IC50 of opaganib and the upamostat active metabolite, WX-UK1 in the HuCCT1 CCA cell line. (B,D) As indicated by an increase in the fluorescence of dead cells, all drug treatments decreased cell viability in CCA cell lines. (C) As indicated by a decrease in cells, WX-UK1 and opaganib in combination inhibit CCA cell migration (scale 300 µm).