Identification of Novel Therapeutic Targets for Fibrolamellar Carcinoma Using Patient-Derived Xenografts and Direct-from-Patient Screening.

To repurpose therapeutics for fibrolamellar carcinoma (FLC), we developed and validated patient-derived xenografts (PDX) from surgical resections. Most agents used clinically and inhibitors of oncogenes overexpressed in FLC showed little efficacy on PDX. A high-throughput functional drug screen found primary and metastatic FLC were vulnerable to clinically available inhibitors of TOPO1 and HDAC and to napabucasin. Napabucasin's efficacy was mediated through reactive oxygen species and inhibition of translation initiation, and specific inhibition of eIF4A was effective. The sensitivity of each PDX line inversely correlated with expression of the antiapoptotic protein Bcl-xL, and inhibition of Bcl-xL synergized with other drugs. Screening directly on cells dissociated from patient resections validated these results. This demonstrates that a direct functional screen on patient tumors provides therapeutically informative data within a clinically useful time frame. Identifying these novel therapeutic targets and combination therapies is an urgent need, as effective therapeutics for FLC are currently unavailable. SIGNIFICANCE: Therapeutics informed by genomics have not yielded effective therapies for FLC. A functional screen identified TOPO1, HDAC inhibitors, and napabucasin as efficacious and synergistic with inhibition of Bcl-xL. Validation on cells dissociated directly from patient tumors demonstrates the ability for functional precision medicine in a solid tumor.This article is highlighted in the In This Issue feature, p. 2355.

Characterization of the full-length human Grb7 protein and a phosphorylation representative mutant.

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways. Extrapolation to the full-length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine-phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated-tyrosine-mimic Y492E-FL-Grb7 protein (Y492E-FL-Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild-type FL Grb7(WT-FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11µM. Our studies here measure a FL protein dimerization Kd of WT-FL-Grb7 within one order of magnitude at approximately 1µM. The approximate size and shape of the WT-FL-Grb7 in comparison the tyrosine-phosphorylation mimic Y492E-FL-Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.

Grb7 protein RA domain oligomerization.

The growth factor receptor bound protein 7 (Grb7) is an adaptor protein that is often coamplified with the erythroblastosis oncogene B 2 receptor in 20% to 30% of breast cancer patients. Grb7 overexpression has been linked to increased cell migration and cancer metastasis. The ras associating and pleckstrin homology domain region of Grb7 has been reported to interact with various other downstream signaling proteins such as four and half Lin11, Isl-1, Mec-3 (LIM) domains isoform 2 and filamin α. These interactions are believed to play a role in regulating Grb7-mediated cell migration function. The full-length Grb7 protein has been shown to dimerize, and the oligomeric state of the Grb7SH2 domain has been extensively studied; however, the oligomerization state of the ras associating and pleckstrin homology domains, and the importance of this oligomerization in Grb7 function, is yet to be fully known. In this study, we characterize the oligomeric state of the Grb7RA domain using size exclusion chromatography, nuclear magnetic resonance, nuclear relaxation studies, glutaraldehyde cross linking, and dynamic light scattering. We report the Grb7RA domain can exist in transient multimeric forms and, based upon modeling results, postulate the potential role of Grb7RA domain oligomerization in Grb7 function.

Grb7 and Hax1 may colocalize partially to mitochondria in EGF-treated SKBR3 cells and their interaction can affect Caspase3 cleavage of Hax1.

Growth factor receptor bound protein 7 (Grb7) is a signal-transducing adaptor protein that mediates specific protein-protein interactions in multiple signaling pathways. Grb7, with Grb10 and Grb14, is members of the Grb7 protein family. The topology of the Grb7 family members contains several protein-binding domains that facilitate the formation of protein complexes, and high signal transduction efficiency. Grb7 has been found overexpressed in several types of cancers and cancer cell lines and is presumed involved in cancer progression through promotion of cell proliferation and migration via interactions with the erythroblastosis oncogene B 2 (human epidermal growth factor receptor 2) receptor, focal adhesion kinase, Ras-GTPases, and other signaling partners. We previously reported Grb7 binds to Hax1 (HS1 associated protein X1) isoform 1, an anti-apoptotic protein also involved in cell proliferation and calcium homeostasis. In this study, we confirm that the in vitro Grb7/Hax1 interaction is exclusive to these two proteins and their interaction does not depend on Grb7 dimerization state. In addition, we report Grb7 and Hax1 isoform 1 may colocalize partially to mitochondria in epidermal growth factor-treated SKBR3 cells and growth conditions can affect this colocalization. Moreover, Grb7 can affect Caspase3 cleavage of Hax1 isoform 1 in vitro, and Grb7 expression may slow Caspase3 cleavage of Hax1 isoform 1 in apoptotic HeLa cells. Finally, Grb7 is shown to increase cell viability in apoptotic HeLa cells in a time-dependent manner. Taken together, these discoveries provide clues for the role of a Grb7/Hax1 protein interaction in apoptosis pathways involving Hax1. Copyright © 2016 John Wiley & Sons, Ltd.

Grb7 and Filamin-a associate and are colocalized to cell membrane ruffles upon EGF stimulation.

Grb7 is an adaptor molecule mediating signal transduction from multiple cell surface receptors to diverse downstream pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase, ErbB2, are overexpressed in 20-30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, although the participants in these pathways have not been fully determined. In this study, we report the Grb7 protein interacts with Filamin-a, an actin-crosslinking component of the cell cytoskeleton. Additionally, we have demonstrated the interaction between Grb7 and Flna is specific to the RA-PH domains of Grb7, and the immunoglobulin-like repeat 16-19 domains of Flna. We demonstrate that full-length Grb7 and Flna interact in the mammalian cellular environment, as well as in vitro. Immunofluorescent microscopy shows potential co-localization of Grb7 and Flna in membrane ruffles upon epidermal growth factor stimulation. These studies are amongst the first to establish a clear connection between Grb7 signaling and cytoskeletal remodeling.

Dimerization in the Grb7 protein.

In previous studies, we showed that the tyrosine phosphorylation state of growth factor receptor-bound protein 7 (Grb7) affects its ability to bind to the transcription regulator FHL2 and the cortactin-interacting protein, human HS-1-associated protein-1. Here, we present results describing the importance of dimerization in the Grb7-Src homology 2 (SH2) domain in terms of its structural integrity and the ability to bind phosphorylated tyrosine peptide ligands. A tyrosine phosphorylation-mimic mutant (Y80E-Grb7-SH2) is largely dimerization deficient and binds a tyrosine-phosphorylated peptide representative of the receptor tyrosine kinase (RTK) erbB2 with differing thermodynamic characteristics than the wild-type SH2 domain. Another dimerization-deficient mutant (F99R-Grb7-SH2) binds the phosphorylated erbB2 peptide with similarly changed thermodynamic characteristics. Both Y80E-Grb7-SH2 and F99R-Grb7-SH2 are structured by circular dichroism measurements but show reduced thermal stability relative to the wild type-Grb7-SH2 domain as measured by circular dichroism and nuclear magnetic resonance. It is well known that the dimerization state of RTKs (as binding partners to adaptor proteins such as Grb7) plays an important role in their regulation. Here, we propose the phosphorylation state of Grb7-SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs such as erbB2. In this manner, additional dimerization-dependent regulation could occur downstream of the membrane-bound kinase in RTK-mediated signaling pathways.

Water-refined solution structure of the human Grb7-SH2 domain in complex with the erbB2 receptor peptide pY1139.

We report a refinement in implicit water of the previously published solution structure of the Grb7-SH2 domain bound to the erbB2 receptor peptide pY1139. Structure quality measures indicate substantial improvement, with residues in the most favored regions of the Ramachandran plot increasing by 14 % and with WHAT IF statistics (Vriend, G. J. Mol. Graph., 1990, 8(1), 52-56) falling closer to expected values for well-refined structures.

Grb7 binds to Hax-1 and undergoes an intramolecular domain association that offers a model for Grb7 regulation.

Adaptor proteins mediate signal transduction from cell surface receptors to downstream signaling pathways. The Grb7 protein family of adaptor proteins is constituted by Grb7, Grb10, and Grb14. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7-mediated cell migration has been shown to proceed through a focal adhesion kinase (FAK)/Grb7 pathway, although the specific participants downstream of Grb7 in cell migration signaling have not been fully determined. In this study, we report that Grb7 interacts with Hax-1, a cytoskeletal-associated protein found overexpressed in metastatic tumors and cancer cell lines. Additionally, in yeast 2-hybrid assays, we show that the interaction is specific to the Grb7-RA and -PH domains. We have also demonstrated that full-length Grb7 and Hax-1 interact in mammalian cells and that Grb7 is tyrosine phosphorylated. Isothermal titration calorimetry measurements demonstrate the Grb7-RA-PH domains bind to the Grb7-SH2 domain with micromolar affinity, suggesting full-length Grb7 can exist in a head-to-tail conformational state that could serve a self-regulatory function.

The intertwining of structure and function: proposed helix-swapping of the SH2 domain of Grb7, a regulatory protein implicated in cancer progression and inflammation.

Grb7 is a multidomain intracellular signaling protein that links activated tyrosine kinases with downstream signaling targets. Best known for its regulatory role in cell migration and tumor metastasis, Grb7 also regulates inflammation by coupling NF-kappaB-inducing kinase with erbB/EGFR family receptors. The "adaptor" role of Grb7 in these processes depends upon binding to membrane-associated tyrosine kinases through its C-terminal SH2 domain. The Grb7-SH2 domain shares structural and functional similarity with the SH2 domain of Grb2, a constituent of the MAP kinase pathway. Both domains show unusual affinity for cyclic (beta-turn) ligands. The Grb2-SH2 domain also shows distinctive self-association behavior, forming intertwined ("swapped") dimers. While Grb7 and its SH2 domain are each known to dimerize, the mechanisms and functional significance of this self-association are incompletely understood. Additional residues in the Grb7-SH2 domain effectively lengthen its "EF loop" and render the domain a good candidate for swapped dimerization, through exchange of a C-terminal helix. We propose the existence of a swapped dimeric form of the Grb7-SH2 domain and offer a structural model derived through novel application of nuclear magnetic resonance-derived restraints.

The cell migration protein Grb7 associates with transcriptional regulator FHL2 in a Grb7 phosphorylation-dependent manner.

Grb7 is an adaptor molecule that can mediate signal transduction from multiple cell surface receptors to various downstream signaling pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase (RTK), erbB2, are overexpressed in 20-30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, though the participants in these pathways have not been determined. In this study, we report that Grb7 interacts with four and half lim domains isoform 2 (FHL2), a transcription regulator with an important role in oncogenesis, including breast cancer. Additionally, in yeast 2-hybrid (Y2H) assays, we show that the interaction is specific to the Grb7 RA and PH domains. We have also demonstrated that full-length (FL) Grb7 and FHL2 interact in mammalian cells and that Grb7 must be tyrosine phosphorylated for this interaction to occur. Immunofluorescent microscopy demonstrates possible co-localization of Grb7 and FHL2. A model with supporting NMR evidence of Grb7 autoinhibition is proposed.

Isolation, characterization, and biological evaluation of syn and anti diastereomers of [(99m)Tc]technetium depreotide: a somatostatin receptor binding tumor imaging agent.

The early and later eluting [(99m)TcO]depreotide products on RP-HPLC were confirmed to be the anti and syn diastereomers, respectively, based on proton NMR and circular dichroism spectroscopy. NMR provided evidence of a folded, conformationally constrained structure for the syn diastereomer. The syn diastereomer is predominant (anti/syn approximately 10:90) in the [(99m)TcO]depreotide preparation and shows a slightly higher affinity (IC50 = 0.15 nM) for the somatostatin receptor than the anti diastereomer (IC50 = 0.89 nM). Both diastereomers showed higher binding affinities than the free peptide (IC(50) = 7.4 nM). Biodistribution studies in AR42J tumor xenograft nude mice also showed higher tumor uptake for syn [(99m)TcO]depreotide (6.58% ID/g) than for the anti [(99m)TcO]depreotide (3.38% ID/g). Despite the differences in biological efficacy, the favorable binding affinity, tumor uptake, and tumor-to-background ratio results for both diastereomeric species predict that both are effective for imaging somatostatin receptor-positive tumors in vivo.

Alternative splicing of the voltage-gated Ca2+ channel beta4 subunit creates a uniquely folded N-terminal protein binding domain with cell-specific expression in the cerebellar cortex.

Ca2+ channel beta subunits regulate cell-surface expression and gating of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, beta subunits are predicted to be modular structures composed of five domains (A-E) that are related to the large family of membrane-associated guanylate kinase proteins. The crystal structure of the beta subunit core B-D domains has been reported recently; however, little is known about the structures of the A and E domains. The N-terminal A domain differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4) primarily as the result of two duplications of an ancestral gene containing multiple alternatively spliced exons. At least nine A domain sequences can be generated by alternative splicing. In this report, we focus on one A domain sequence, the highly conserved beta4a A domain. We solved its three-dimensional structure and show that it is expressed in punctate structures throughout the molecular layer of the cerebellar cortex. We also demonstrate that it does not participate directly in Cav2.1 Ca2+ channel gating but serves as a binding site in protein-protein interactions with synaptotagmin I and the LC2 domain of microtubule-associated protein 1A. With respect to beta4 subunits, the interactions are specific for the beta4a splice variant, because they do not occur with the beta4b A domain. These results have strong bearing on our current understanding of the structure of alternatively spliced Ca2+ channel beta subunits and the cell-specific roles they play in the CNS.

Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit.

Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating.

Backbone nuclear relaxation characteristics and calorimetric investigation of the human Grb7-SH2/erbB2 peptide complex.

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20%-30% of breast cancers. Grb7 binds to erbB2 and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In a prior study, we reported the solution structure of the Grb7-SH2/erbB2 peptide complex. In this study, T(1), T(2), and steady-state NOE measurements were performed on the Grb7-SH2 domain, and the backbone relaxation behavior of the domain is discussed with respect to the potential function of an insert region present in all three members of this protein family. Isothermal titration calorimetry (ITC) studies were completed measuring the thermodynamic parameters of the binding of a 10-residue phosphorylated peptide representative of erbB2 to the SH2 domain. These measurements are compared to calorimetric studies performed on other SH2 domain/phosphorylated peptide complexes available in the literature.

Solution structure of the human Grb14-SH2 domain and comparison with the structures of the human Grb7-SH2/erbB2 peptide complex and human Grb10-SH2 domain.

Grb14 is an adapter protein that is known to be overexpressed in estrogen receptor positive breast cancers, and in a number of prostate cancer cell lines. Grb14 has been demonstrated to bind to a number of activated receptor tyrosine kinases (RTKs) and to modulate signals transduced through these receptors. The RTKs to which Grb14 binds include the insulin receptor (IR), the fibroblast growth factor receptor (FGFR), the platelet-derived growth factor receptor (PDGFR), and the tunica endothelial kinase (Tek/Tie2) receptor. Grb14 has been shown to bind to these activated RTKs through its Src homology 2 (SH2) domain, with the exception of the insulin receptor, where the primary binding interaction is via a small domain adjacent to the SH2 domain (the BPS or PIR domain). Grb14 is a member of the Grb7 family of proteins, which also includes Grb7 and Grb10. We have solved the solution structure of the human Grb14-SH2 domain and compared it with the recently determined Grb7-SH2 and Grb10-SH2 domain structures.

Solution structure of the human Grb7-SH2 domain/erbB2 peptide complex and structural basis for Grb7 binding to ErbB2.

The solution structure of the hGrb7-SH2 domain in complex with a ten amino acid phosphorylated peptide ligand representative of the erbB2 receptor tyrosine kinase (pY1139) is presented as determined by nuclear magnetic resonance methods. The hGrb7-SH2 domain structure reveals the Src homology 2 domain topology consisting of a central beta-sheet capped at each end by an alpha-helix. The presence of a four residue insertion in the region between beta-strand E and the EF loop and resulting influences on the SH2 domain/peptide complex structure are discussed. The binding conformation of the erbB2 peptide is in a beta-turn similar to that found in phosphorylated tyrosine peptides bound to the Grb2-SH2 domain. To our knowledge this is only the second example of an SH2 domain binding its naturally occurring ligands in a turn, instead of extended, conformation. Close contacts between residues responsible for binding specificity in hGrb7-SH2 and the erbB2 peptide are characterized and the potential effect of mutation of these residues on the hGrb7-SH2 domain structure is discussed.

Assignment of backbone 1H, 13C, and 15N resonances of the SH2 domain of human Grb14.

The (1)H, (13)C, and (15)N backbone resonance assignments have been made for the Src homology 2 (SH2) domain of the human molecular adapter protein Grb14. The assignments, along with the majority of the non-aromatic side-chain (1)H and (13)C resonances are reported. The SH2 domain has been complexed with a phosphotyrosine-containing peptide (pY766) corresponding to the putative binding site in the fibroblast growth factor receptor (FGFR1). Chemical shift changes upon binding are also reported.