Martyna Sochacka

FGF12 belongs to a subfamily of FGF proteins called FGF homologous factors (FHFs), which until recently were thought to be non-signaling intracellular proteins. Our recent studies have shown that although they lack a conventional signal peptide for secretion, they can reach the extracellular space, especially under stress conditions. Here, we unraveled that the long “a” isoform of FGF12 is secreted in a pathway involving the A1 subunit of Na(+)/K(+) ATPase (ATP1A1), Tec kinase and lipids such as phosphatidylinositol and phosphatidylserine. Further, we showed that the short “b” isoform of FGF12, which binds ATP1A1 and phosphatidylserine less efficiently, is not secreted from cells. We also indicated regions in the FGF12a protein sequence that are crucial for its secretion, including N-terminal fragment and specific residues, and proposed that liquid-liquid phase separation may be important in this process. Our results strongly suggest that the mechanism of this process is very similar for all unconventionally secreted FGF proteins.

FGF homologous factors (FHFs) are the least described group of fibroblast growth factors (FGFs). The FHF subfamily consists of four proteins: FGF11, FGF12, FGF13, and FGF14. Until recently, FHFs were thought to be intracellular, non‐signaling molecules, despite sharing structural and sequence similarities with other members of FGF family that can be secreted and activate cell signaling by interacting with surface receptors. Here, we show that despite lacking a canonical signal peptide for secretion, FHFs are exported to the extracellular space. Furthermore, we propose that their secretion mechanism is similar to the unconventional secretion of FGF2. The secreted FHFs are biologically active and trigger signaling in cells expressing FGF receptors (FGFRs). Using recombinant proteins, we demonstrated their direct binding to FGFR1, resulting in the activation of downstream signaling and the internalization of the FHF‐FGFR1 complex. The effect of receptor activation by FHF proteins is an anti‐apoptotic response of the cell.

Among the FGF proteins, the least characterized superfamily is the group of fibroblast growth factor homologous factors (FHFs). To date, the main role of FHFs has been primarily seen in the modulation of voltage-gated ion channels, but a full picture of the function of FHFs inside the cell is far from complete. In the present study, we focused on identifying novel FGF12 binding partners to indicate its intracellular functions. Among the identified proteins, a significant number were nuclear proteins, especially RNA-binding proteins involved in translational processes, such as ribosomal processing and modification. We have demonstrated that FGF12 is localized to the nucleolus, where it interacts with NOLC1 and TCOF1, proteins involved in the assembly of functional ribosomes. Interactions with both NOLC1 and TCOF1 are unique to FGF12, as other FHF proteins only bind to TCOF1. The formation of nucleolar FGF12 complexes with NOLC1 and TCOF1 is phosphorylation-dependent and requires the C-terminal region of FGF12. Surprisingly, NOLC1 and TCOF1 are unable to interact with each other in the absence of FGF12. Taken together, our data link FHF proteins to nucleoli for the first time and suggest a novel and unexpected role for FGF12 in ribosome biogenesis.

Background: Antibody drug conjugates (ADCs) represent one of the most promising approaches in the current immuno-oncology research. The precise delivery of cytotoxic drugs to the cancer cells using ADCs specific for tumor-associated antigens enables sparing the healthy cells and thereby reduces unwanted side effects. Overexpression of fibroblast growth factor receptor 1 (FGFR1) has been demonstrated in numerous tumors and thereby constitutes a convenient molecular target for selective cancer treatment. We have recently engineered tetravalent anti-FGFR1 antibody, T-Fc, and have demonstrated that it displays extremely efficient internalization into FGFR1 producing cells, a feature highly desirable in the ADC approach. We have revealed that T-Fc mediates clustering of FGFR1, largely enhancing the uptake of FGFR1-T-Fc complexes by induction of clathrin-independent endocytic routes. The aim of this study was to obtain highly internalizing cytotoxic conjugate of the T-Fc for specific delivery of drugs into FGFR1-positive cancer cells. Methods: Conjugation of the T-Fc to a cytotoxic payload, vcMMAE, was carried out via maleimide chemistry, yielding the T-Fc-vcMMAE. The specific binding of the T-Fc-vcMMAE conjugate to FGFR1 was confirmed in vitro with BLI technique. Confocal microscopy and flow cytometry were applied to determine FGFR1-dependence of the T-Fc-vcMMAE internalization. Western blot analyses of FGFR1-dependent signaling were conducted to assess the impact of the T-Fc-vcMMAE on FGFR1 activation and initiation of downstream signaling cascades. Finally, using FGFR1-negative and FGFR1-possitive cell lines, the cytotoxic potential of the T-Fc-vcMMAE was evaluated. Results: We have performed the efficient conjugation of the tetravalent engineered antibody with a cytotoxic drug and generated FGFR1-specific ADC molecule, T-Fc-vcMMAE. We have demonstrated that T-Fc-vcMMAE conjugate exhibits high selectivity and affinity for FGFR1, similarly to T-Fc. Furthermore, we have shown that T-Fc constitutes an effective drug delivery vehicle as T-Fc-vcMMAE was efficiently and selectively internalized by FGFR1-producing cells leading to their death. Interestingly, we show that the efficiency of the uptake of T-Fc-vcMMAE corresponds well with the cytotoxicity of the conjugate, but doesn’t correlate with the FGFR1expression level. Conclusion: Our results show that T-Fc-vcMMAE fulfills the key criteria for the successful cytotoxic drug carrier in a targeted approach against FGFR1-positive cancer cells. Furthermore, our data implicate that not solely expression level of the receptor, but rather its cellular trafficking should be taken into account for selection of suitable molecular targets and cancer models for successful ADC approach.

Fibroblast growth factors (FGFs) via their receptors (FGFRs) transduce signals from the extracellular space to the cell interior, modulating pivotal cellular processes such as cell proliferation, motility, metabolism and death. FGF superfamily includes a group of fibroblast growth factor homologous factors (FHFs), proteins whose function is still largely unknown. Since FHFs lack the signal sequence for secretion and are unable to induce FGFR-dependent cell proliferation, these proteins were considered as intracellular proteins that are not involved in signal transduction via FGFRs. Here we demonstrate for the first time that FHF1 directly interacts with all four major FGFRs. FHF1 binding causes efficient FGFR activation and initiation of receptor-dependent signaling cascades. However, the biological effect of FHF1 differs from the one elicited by canonical FGFs, as extracellular FHF1 protects cells from apoptosis, but is unable to stimulate cell division. Our data define FHF1 as a FGFR ligand, emphasizing much greater similarity between FHFs and canonical FGFs than previously indicated. Graphical

Fibroblast growth factor 1 (FGF1) has been shown to interact with integrin α_vβ₃ through a specific binding site, involving Arg35 residue. The FGF1 mutant (R35E) with impaired integrin binding was found to be defective in its proliferative response, although it was still able to interact with FGF receptors (FGFR) and heparin and induce the activation of downstream signaling pathways. Here, we demonstrate that the lack of mitogenic potential of R35E mutant is directly caused by its decreased thermodynamic stability and susceptibility to proteolytic degradation. Introduction of three stabilizing mutations into R35E variant compensated the effect of destabilizing R35E mutation and restored the proliferation potential of FGF1. Moreover, the stabilized R35E variant regained both anti-apoptotic and wound healing activities, while remaining defective in binding to integrin α_vβ₃. Our results suggest that the thermodynamic stability and resistance to degradation, rather than the interaction with integrin are required for mitogenic response of FGF1.

Fibroblast growth factor receptor 1 (FGFR1) is a plasma membrane protein that transmits signals from the extracellular environment, regulating cell homeostasis and function. Dysregulation of FGFR1 leads to the development of human cancers and noncancerous diseases. Numerous tumors overproduce FGFR1, making this receptor a perspective target for cancer therapies. Antibody-drug conjugates (ADCs) are highly potent and selective anticancer agents. ADCs are composed of antibodies (targeting factors) fused to highly cytotoxic drugs (warheads). The efficiency of ADC strategy largely depends on the internalization of cytotoxic conjugate into cancer cells. Here, we have studied an interplay between affinity of anti-FGFR1 antibodies and efficiency of their cellular uptake. We have developed a unique set of engineered anti-FGFR1 antibodies that bind the same epitope in the extracellular part of FGFR1, but with different affinities. We have demonstrated that these antibodies are effectively taken up by cancer cells in the FGFR1-dependent manner. Interestingly, we have found that efficiency, defined as rate and level of antibody internalization, largely depends on the affinity of engineered antibodies towards FGFR1, as high affinity antibody displays fastest internalization kinetics. Our data may facilitate design of therapeutically relevant targeting molecules for selective treatment of FGFR1 overproducing cancers.