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Mutations in FGFR2 are common in a subset of endometrial carcinomas. Given the emergence of small molecule inhibitors specific to this receptor tyrosine kinase, FGFR2 is an attractive therapeutic target. However, compensatory and adaptation mechanisms limit the clinical utility of compounds that target nodes in the receptor tyrosine kinase network. Here, we analysed the impact of FGFR inhibition in endometrial cancer cells and observed the emergence of a resistant population in an FGFR2-mutant cell line. To understand the mechanisms underlying this adaptation response, we used a phosphoproteomics approach to measure the kinase network in an unbiased manner. These experiments led to the identification of an AKT-related compensatory mechanism underpinning this resistance. Further dissection of this resistance mechanism utilising gene expression analysis showed PHLDA1, a negative regulator of AKT, was significantly down-regulated in resistant cells. This was further confirmed at the protein level. siRNA knockdown of PHLDA1 conferred immediate drug resistance in the FGFR2-mutant endometrial cancer cell line. Therefore, we identified PHLDA1 down-regulation as a mediator of drug resistance in FGFR2 mutant cancer cells, the first demonstration of the role of PHLDA1 in the acquisition and maintenance of drug resistance. Using a 3D physiomimetic model, we demonstrated that AKT inhibition alone also led to generation of a drug-resistant population. Most importantly, dual-drug therapy inhibited proliferation and induced cell death. Our data highlight how mass spectrometry and microarray gene expression analysis can complement each other in the identification of novel resistance mechanisms in cancer cells. These data suggest that combination treatment of FGFR2-mutant endometrial cancers, targeting both FGFR2 and AKT, represents a promising therapeutic approach.

Fibroblast growth factor receptor plays a major role in several biological processes. Without FGFR, a human cannot live. FGFR is involved in cell differentiation and wound healing. Of course, if FGFR signaling becomes unregulated, it causes severe distress in the body. Several cancers are contributed to high signaling levels, as well as developmental conditions like rickets and Kallmann’s syndrome. FGFR is thought to undergo an auto-inhibition (or self-regulatory) process in order to try to facilitate regulation. The exact method of this inhibition is currently unknown, but is proposed to involve the unstructured acid box region of FGFR. We developed a simple model system in order to further investigate current models of inhibition that FGFR may undergo. By using our model system, which contains two 15-mer homopolypeptides of polyE and polyK that mimic the acid box region and its binding site respectively, we were able to use a combination of ITC, CD, NMR, and FRET to show that one model from the literature contains flaws. We are able to characterize the binding of our polypeptide system under varying ionic conditions and pH. This model system also provides a platform to better understand general principles of charge-charge interactions in proteins, which are often characterized by FRET. One of the important findings from this study is that 15-mers of polyE and polyK bind in a parallel arrangement. One of the hurdles in applying FRET to such systems is determining the role that the attached FRET dyes play in the charge-charge interactions. Our model system allowed us to use the preference of the charged polypeptides to bind in a parallel arrangement to determine the size, charge and structural effects of the attached FRET dyes on how peptides bind under electrostatic interaction conditions and to quantify how the attached fluorescent dyes are quenched by both the charged amino acids as well as by the FRET acceptor.

Fibroblast Growth Factor Receptors (FGFRs) are critical for cell proliferation and differentiation. Mutation and/or translocation of FGFRs lead to aberrant signaling that often results in developmental syndromes or cancer growth. As sequencing of human tumors becomes more frequent, so does the emergence of FGFR translocations and fusion proteins. The research conducted in this work will focus on a frequently identified fusion protein between FGFR3 and transforming acidic coiled-coil containing protein 3 (TACC3). Through titanium dioxide-based phosphopeptide enrichment (TiO2)-liquid chromatography (LC)-high mass accuracy tandem mass spectrometry (MS/MS), it is apparent that the fused coiled-coil TACC3 domain results in constitutive phosphorylation of key activating FGFR3 tyrosine residues. Fusion of FGFR3 and TACC3 also results in MAPK pathway activation, nuclear localization, and cell transformation. Introduction of K508R FGFR3 kinase dead mutation abrogates these effects, except for nuclear localization which is due solely to the TACC3 domain. FGFR3-TACC3 also produces IL-3 independent growth and cell proliferation. Taken together, these results suggest that FGFR3 kinase activity is essential for the oncogenic effects of the FGFR3-TACC3 fusion protein and could serve as a therapeutic target.

Cancer is a set of complex genetic diseases driven by diverse genomic alterations. The genomic study of cancer has enabled the discovery of novel, targetable events in almost all cancer types and in turn, has led to the development of new, targeted cancer therapies benefiting patients; however, the recent explosion of genomic datasets has also resulted in huge lists of new oncogenic factors of unknown biological relevance, and uncertainty over how best to use the data appropriately to influence patient care. Some of the most pressing questions surround the use of statistical methods to identify actionable genomic alterations in cancer and the identification of driving oncogenes in the context of the genomic evolution of cancer cells, undergone before, during, and after prolonged treatment regimens.

The interactions between FGF and fibroblast growth factor receptors are responsible for the regulation of key cellular processes. FGF is important in both germ cell and embryonic developments. FGF continues to play important roles during adulthood by regulating embryogenesis, cell differentiation, and wound healing (1-7). The regulations of these cellular events are initiated through FGF binding to the fibroblast growth factor receptors. The complex formed by FGF and the receptor involves a key interaction with heparin. Through interactions with heparin, the FGF, FGFR and Heparin form a 2:2:2 complex (8). This complex formation results in autophosphorylation in the tyrosine kinase domain in the cytoplasm. The autophosphorylation events lead to downstream signaling that result in the regulation of previously mentioned cellular processes (9, 10). Mutations within the FGF or FGFR may interfere with signaling or protein stability. Changes in the signaling efficiency by FGF or the FGFR are shown to lead to disease states There exist many point mutations in the FGF receptor that result in craniofacial, hypogonadotropic hypogonadism, anosmia, and tumor development. Using site-directed mutagenesis we have shown non-covalent interactions formed by Kallmann syndrome linked mutations result in a loss-of-binding between FGF and the FGF receptor. This evidence has shown that the non-covalent ligand binding interactions lost are due to changes in the D2 structure or binding site. Additionally, the R203C mutation, linked to breast cancer, was tested and determined to break a D2 stabilizing cation-ð bond. The cation-ð stabilized the binding interaction with heparin and provides stability to the D2 domain. Although the decreased stability of the D2 domain supports a loss-of-function, we are currently investigating intermolecular disulfide bond formation between adjacent receptor. This is a known mechanism among FGF receptors that may lead to signaling in the absence of a ligand.