Rhenium N-heterocyclic carbene complexes block growth of aggressive cancers by inhibiting FGFR- and SRC-mediated signalling

Figures

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  Fig. 1. 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)

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  Fig. 2. 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

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  Fig. 3. 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

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  Fig. 4. 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

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  Fig. 5. 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

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  Fig. 6. 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

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  Fig. 7. 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)