Mechanism of action of tricarbonyl rhenium complexes.a Summary of SelectScreen Kinase Profiling showing the % inhibition of JVG045 and ps27 on the kinases FGFR1 and Src. b Western blots in pancreatic cancer cell lines AsPC-1 and HPAF-II showing that JVG045 (10 μM) indeed inhibits the phosphorylation of FGFR1 with an effect comparable to the selective FGFR inhibitor BGJ398 (5 μM); ReCl was used as negative control. c Autophosphorylation activity of FGFR1, no difference in kinase activity is detected in the presence of JVG045, indicating that there is no direct inhibition. d Src kinase assay shows that, when used on the purified kinase, JVG045 does not have a direct action compared to the selective Src inhibitor AZD0530. e, f JVG045 (10 μM) increases the production of reactive oxygen species (ROS) in pancreatic cancer cell lines AsPC-1 (e) and HPAF-II (f) measured using the H2DCF-DA probe. Results are expressed as Mean ± SEM and are representative of at least three independent experiments. Mean fluorescence intensity DMSO: AsPC1 = 5368 + 1517 (n = 4); HPAF-II =4094 + 1454 (n = 3)

In vivo toxicity of tricarbonyl rhenium complexes JVG045 and ps27. (A-H) Graphs comparing the effect of a dose response of JVG045, ps27, Cisplatin and DMSO control on zebrafish hatching (a-d) and embryo mortality (e-h) from 24 h post fertilisation (hpf) until 120hpf. i Effect of a dose response of JVG045 on zebrafish heartbeats showing no significant difference up to 500 μM compared to DMSO. j BGJ398 is a competitive FGFR1–3 inhibitor [38, 39] acting on multiple FGFRs in the zebrafish embryo and causing developmental defects starting at concentrations as low as 0.5-1 μM. On the other hand, as a putative FGFR1 inhibitor, JVG045 does not cause developmental defects in the zebrafish embryo. Treatment started at 2hpf and images were taken at 24hpf. Results are showed as Mean ± SEM and are representative of at least three independent experiments

Effect of JVG045 on primary pancreatic cancer cells from the KPC mouse model.a Representative images showing the effect of JVG045 at 10 μM on the number of primary pancreatic cancer cells isolated from the KrasLSL.G12D/+; p53R172H/+; PdxCretg/+ (or KPC) mouse model, compared to DMSO. b Graph showing a dose response effect of JVG045 on KPC cells, with 5 and 10 μM being the most effective concentrations. c Representative mages showing the effect of JVG045 (10 μM) on anchorage-independent colony formation of KPC cells, compared to DMSO. d Graph showing a significant reduction in KPC colonies formed in the presence of JVG045 (10 μM), compared to DMSO. Experiments were performed in triplicate and showed as Mean ± SEM

Effect of JVG045 on human neuroblastoma cell lines.a Dose response of JVG045 on human neuroblastoma cell lines. The graph shows that the tricarbonyl Rhenium compound has very little effect on cell number in those cell lines with non-amplified MYCN (SHSY5 and hNB), while it has a significant effect in MYCN-amplified cell lines (Kelly, IMR-32 and LAN-1). b Representative western blot showing the effect of JVG045 (10 μM) on the phosphorylation of FGFR1(Y653/654) and Src(Y416) compared to the FGFR inhibitor BGJ398 and the Src inhibitor Bosutinib, respectively. ReCl was used as a negative control. Data from R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl) databases illustrating the prognostic value of FGFR1 and Src in Pancreatic Ductal Adenocarcinoma (PDAC) (a and e), and Neuroblastoma (d and f). Results are showed as Mean ± SEM and experiments were performed in triplicate

Effect of JVG045 on pancreatic cancer cell lines depends on mutationally activated KRAS.a Graph showing that in the BxPC-3 human pancreatic cancer cell line (which lacks a KRAS mutation) increasing doses of JVG045 had no significant effect on cell number. In contrast, SW1990, a pancreatic cancer cell line bearing a KRAS mutation and wt P53, showed enhanced sensitivity to increasing concentrations of JVG045. b Representative western blot showing the effect of JVG045 and ps27 (both at 10 μM) on the phosphorylation of FGFR1(Y653/654) and Src(Y416) in the presence or absence of the FGF ligand (20 ng/ml). ReCl was used as a negative control. c Effect of JVG045 (10 μM) in significantly reducing cell numbers in chemoresistant AsPC-1 tumorspheres enriched in cancer stem cells. d JVG045 (10 μM) shows no significant effect in KRAS wt BxPC-3 tumorspheres. e Effect of JVG045 (10 μM) in significantly reducing cell number in chemoresistant primary KPC tumorspheres enriched in cancer stem cells. Results are expressed as Mean ± SEM and are representative of at least three independent experiments

Effect of JVG045 on zebrafish and mouse xenografts.a Brightfield images and images showing DiL-labelled HPAF-II tumour xenografts in 5 days post-fertilisation zebrafish embryos untreated (DMSO) or treated with JVG045 (10 μM) for 3 days after injection of HPAF-II human pancreatic cancer cells. Scale bar 100 μm. b Graph showing the effect of 10 μM JVG045 in reducing the tumour burden in zebrafish embryos (n = 12), compared to control embryos (DMSO, n = 6). c Representative images of HPAF-II tumours from mouse xenografts receiving control treatment (vehicle) or treated with JVG045 (30mh/kg) for 25 days. d Graph showing tumour growth of HPAF-II xenograft mouse models treated with vehicle (n = 8) and 30 mg/kg JVG045 (n = 9). e Graph showing the tumour weight at the end of the experiment of HPAF-II xenograft mouse models treated with vehicle (n = 8) and 30 mg/kg JVG045 (n = 9). Results are expressed as Mean ± SEM

Effect of JVG045 on FGFR1 and Src in HPAF-II mouse xenografts.a Representative western blot of two groups of tumours either vehicle treated (ctrl) or treated with JVG045 (30 mg/kg) and probed for pSrc (Y416) or pFGFR1(Y653/654). GAPDH was used as loading control. Averaged quantification of (b) pSrc(Y416) and (c) pFGFR1(Y653/654) western blot signals, normalised to their loading control in vehicle treated tumour xenografts (control) or tumour xenografts treated with JVG045 (30 mg/kg)