Strategies to inhibit FGFR4 V550L-driven rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is a paediatric cancer driven either by fusion proteins (e.g., PAX3-FOXO1) or by mutations in key signalling molecules (e.g., RAS or FGFR4). Despite the latter providing opportunities for precision medicine approaches in RMS, there are currently no such treatments implemented in the clinic. METHODS: We evaluated biologic properties and targeting strategies for the FGFR4 V550L activating mutation in RMS559 cells, which have a high allelic fraction of this mutation and are oncogenically dependent on FGFR4 signalling. Signalling and trafficking of FGFR4 V550L were characterised by confocal microscopy and proteomics. Drug effects were determined by live-cell imaging, MTS assay, and in a mouse model. RESULTS: Among recently developed FGFR4-specific inhibitors, FGF401 inhibited FGFR4 V550L-dependent signalling and cell proliferation at low nanomolar concentrations. Two other FGFR4 inhibitors, BLU9931 and H3B6527, lacked potent activity against FGFR4 V550L. Alternate targeting strategies were identified by RMS559 phosphoproteomic analyses, demonstrating that RAS/MAPK and PI3K/AKT are essential druggable pathways downstream of FGFR4 V550L. Furthermore, we found that FGFR4 V550L is HSP90-dependent, and HSP90 inhibitors efficiently impeded RMS559 proliferation. In a RMS559 mouse xenograft model, the pan-FGFR inhibitor, LY2874455, did not efficiently inhibit growth, whereas FGF401 potently abrogated growth. CONCLUSIONS: Our results pave the way for precision medicine approaches against FGFR4 V550L-driven RMS.

ALFY localizes to early endosomes and cellular protrusions to facilitate directional cell migration.

Cell migration is a complex process underlying physiological and pathological processes such as brain development and cancer metastasis. The autophagy-linked FYVE protein (ALFY; also known as WDFY3), an autophagy adaptor protein known to promote clearance of protein aggregates, has been implicated in brain development and neural migration during cerebral cortical neurogenesis in mice. However, a specific role of ALFY in cell motility and extracellular matrix adhesion during migration has not been investigated. Here, we reveal a novel role for ALFY in the endocytic pathway and in cell migration. We show that ALFY localizes to RAB5- and EEA1-positive early endosomes in a PtdIns(3)P-dependent manner and is highly enriched in cellular protrusions at the leading and lagging edge of migrating cells. We find that cells lacking ALFY have reduced attachment and altered protein levels and glycosylation of integrins, resulting in the inability to form a proper leading edge and loss of directional cell motility.

Fibroblast Growth Factor 2 Conjugated with Monomethyl Auristatin E Inhibits Tumor Growth in a Mouse Model.

Worldwide, cancer is the second leading cause of death. Regardless of the continuous progress in medicine, we still do not have a fully effective anti-cancer therapy. Therefore, the search for new targeted anti-cancer drugs is still an unmet need. Here, we present novel protein-drug conjugates that inhibit tumor growth in a mouse model of human breast cancer. We developed conjugates based on fibroblast growth factor (FGF2) with improved biophysical and biological properties for the efficient killing of cancer cells overproducing fibroblast growth factor receptor 1 (FGFR1). We used hydrophilic and biocompatible PEG4 or PEG27 molecules as a spacer between FGF2 and the toxic agent monomethyl auristatin E. All conjugates exhibited a cytotoxic effect on FGFR1-positive cancer cell lines. The conjugate with the highest hydrodynamic size (42 kDa) and cytotoxicity was found to efficiently inhibit tumor growth in a mouse model of human breast cancer.

Crosstalk between p38 and Erk 1/2 in Downregulation of FGF1-Induced Signaling.

Mitogen-activated protein kinases (MAPK): Erk1 and Erk2 are key players in negative-feedback regulation of fibroblast growth factor (FGF) signaling. Upon activation, Erk1 and Erk2 directly phosphorylate FGF receptor 1 (FGFR1) at a specific serine residue in the C-terminal part of the receptor, substantially reducing the tyrosine phosphorylation in the receptor kinase domain and its signaling. Similarly, active Erks can also phosphorylate multiple threonine residues in the docking protein FGF receptor substrate 2 (FRS2), a major mediator of FGFR signaling. Here, we demonstrate that in NIH3T3 mouse fibroblasts and human osteosarcoma U2OS cells stably expressing FGFR1, in addition to Erk1 and Erk2, p38 kinase is able to phosphorylate FRS2. Simultaneous inhibition of Erk1/2 and p38 kinase led to a significant change in the phosphorylation pattern of FRS2 that in turn resulted in prolonged tyrosine phosphorylation of FGFR1 and FRS2 and in sustained signaling, as compared to the selective inhibition of Erks. Furthermore, excessive activation of p38 with anisomycin partially compensated the lack of Erks activity. These experiments reveal a novel crosstalk between p38 and Erk1/2 in downregulation of FGF-induced signaling.

PIKfyve, MTMR3 and their product PtdIns5P regulate cancer cell migration and invasion through activation of Rac1.

Previously, we have shown that the phosphoinositide metabolizing enzymes PIKfyve (phosphoinositide 5-kinase, FYVE finger containing) and MTMR3 (myotubularin-related protein 3), together with their lipid product PtdIns5P, are important for migration of normal human fibroblasts. As these proteins are a kinase and a phosphatase respectively, and thereby considered druggable, we wanted to test their involvement in cancer cell migration and invasion. First, we showed that PIKfyve and MTMR3 are expressed in most cancer cells. Next, we demonstrated that depletion of PIKfyve or MTMR3 resulted in decreased velocity in three different cancer cell lines by using new software for cell tracking. Inhibition of the enzymatic activity of PIKfyve by the inhibitor YM201636 also led to a strong reduction in cell velocity. Mechanistically, we show that PIKfyve and MTMR3 regulate the activation of the Rho family GTPase Rac1. Further experiments also implicated PtdIns5P in the activation of Rac1. The results suggest a model for the activation of Rac1 in cell migration where PIKfyve and MTMR3 produce PtdIns5P on cellular membranes which may then serve to recruit effectors to activate Rac1. Finally, in an invasion assay, we demonstrate that both PIKfyve and MTMR3 are implicated in invasive behaviour of cancer cells. Thus PIKfyve and MTMR3 could represent novel therapeutic targets in metastatic cancer.

Photoactivation approaches reveal a role for Rab11 in FGFR4 recycling and signalling.

Fibroblast growth factor receptor 4 (FGFR4) plays important roles during development and in the adult to maintain tissue homeostasis. Moreover, overexpression of FGFR4 or activating mutations in FGFR4 has been identified as tumour-promoting events in several forms of cancer. Endocytosis is important for regulation of signalling receptors and we have previously shown that FGFR4 is mainly localized to transferrin-positive structures after ligand-induced endocytosis. Here, using a cell line with a defined pericentriolar endocytic recycling compartment, we show that FGFR4 accumulates in this compartment after endocytosis. Furthermore, using classical recycling assays and a new, photoactivatable FGFR4-PA-GFP fusion protein combined with live-cell imaging, we demonstrate that recycling of FGFR4 is dependent on Rab11. Upon Rab11b depletion, FGFR4 is trapped in the pericentriolar recycling compartment and the total levels of FGFR4 in cells are increased. Moreover, fibroblast growth factor 1 (FGF1)-induced autophosphorylation of FGFR4 as well as phosphorylation of phospholipase C (PLC)-γ is prolonged in cells depleted of Rab11. Interestingly, the activation of mitogen-activated protein kinase and AKT pathways were not prolonged but rather reduced in Rab11-depleted cells, indicating that recycling of FGFR4 is important for the nature of its signalling output. Thus, Rab11-dependent recycling of FGFR4 maintains proper levels of FGFR4 in cells and regulates FGF1-induced FGFR4 signalling.

Production of phosphatidylinositol 5-phosphate via PIKfyve and MTMR3 regulates cell migration.

Although phosphatidylinositol 5-phosphate (PtdIns5P) is present in many cell types and its biogenesis is increased by diverse stimuli, its precise cellular function remains elusive. Here we show that PtdIns5P levels increase when cells are stimulated to move and we find PtdIns5P to promote cell migration in tissue culture and in a Drosophila in vivo model. First, class III phosphatidylinositol 3-kinase, which produces PtdIns3P, was shown to be involved in migration of fibroblasts. In a cell migration screen for proteins containing PtdIns3P-binding motifs, we identified the phosphoinositide 5-kinase PIKfyve and the phosphoinositide 3-phosphatase MTMR3, which together constitute a phosphoinositide loop that produces PtdIns5P via PtdIns(3,5)P(2). The ability of PtdIns5P to stimulate cell migration was demonstrated directly with exogenous PtdIns5P and a PtdIns5P-producing bacterial enzyme. Thus, the identified phosphoinositide loop defines a new role for PtdIns5P in cell migration.

Nuclear import of exogenous FGF1 requires the ER-protein LRRC59 and the importins Kpnα1 and Kpnß1.

Fibroblast growth factor 1 (FGF1) taken up by cells into endocytic vesicles can be translocated across vesicular membranes into the cytosol and the nucleus where it has a growth regulatory activity. Previously, leucine-rich repeat containing 59 (LRRC59) was identified as an intracellular binding partner of FGF1, but its biological role remained unknown. Here, we show that LRRC59 is strictly required for nuclear import of exogenous FGF1. siRNA-mediated depletion of LRRC59 did not inhibit the translocation of FGF1 into cytosol, but blocked the nuclear import of FGF1. We also found that an nuclear localization sequence (NLS) in FGF1, Ran GTPase, karyopherin-α1 (Kpnα1), and Kpnß1 were required for nuclear import of FGF1. Nuclear import of exogenous FGF2, which depends on CEP57/Translokin, was independent of LRRC59, but was dependent on Kpnα1 and Kpnß1, while the nuclear import of FGF1 was independent of CEP57. LRRC59 is a membrane-anchored protein that localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE). We found that LRRC59 possesses NLS-like sequences in its cytosolic part that can mediate nuclear import of soluble LRRC59 variants, and that the localization of LRRC59 to the NE depends on Kpnß1. We propose that LRRC59 facilitates transport of cytosolic FGF1 through nuclear pores by interaction with Kpns and movement of LRRC59 along the ER and NE membranes.