Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19–29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15–25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19–29 S20G may serve as a model for the toxic spine of hIAPP.DOI:http://dx.doi.org/10.7554/eLife.19273.001 In Type-II Diabetes, an individual’s cells fail to respond correctly to the hormone insulin, leaving them unable to counteract high levels of sugar in the blood. Another hormone, human islet amyloid polypeptide (hIAPP), works with insulin to regulate blood sugar levels. hIAPP is an amyloid protein, which means that it can lose its normal structure and form fibrils. Fibrils are difficult for cells to break down and are often associated with disease. Indeed, fibrils of hIAPP often form in the pancreas as part of Type-II Diabetes.Some studies have shown that hIAPP fibrils are toxic to pancreatic cells and worsen the symptoms of Type-II Diabetes. Others suggest that it is the process of fibril formation that is toxic, not the fibrils themselves. Although the structures of the fibrils have been described, whether these structures cause cell toxicity has not been investigated.Krotee et al. have now explored the structures of two overlapping segments of hIAPP using a new cryo electron microscopy method called MicroED that is ideal for studying such segments. One segment, called 19-29 S20G, forms a standard amyloid fibril structure that is similar to the structure of full-length hIAPP fibrils. Adding these segments to human cells causes similar levels of toxicity as the full-length hIAPP fibrils. The second segment, called 15-25 WT, forms a non-toxic structure that is less stable than standard amyloid fibrils.The results presented by Krotee et al. support the view that standard amyloid fibril structures are toxic to cells and suggest that 19-29 S20G may be a good model to use when studying how full-length hIAPP fibrils behave. The structure of 19-29 S20G may also be useful as a template for designing molecules that block amyloid fibril growth. If amyloid fibrils cause cell toxicity in the pancreas, then these molecules could be used to treat Type-II Diabetes.DOI:http://dx.doi.org/10.7554/eLife.19273.002 Amyloid fibrils are associated with more than 25 diseases, including Alzheimer’s disease, Parkinson’s disease, and Type-II Diabetes (T2D) . The fibResearchers have accumulated substantial evidence for a correlation between hIAPP aggregation and pancreatic β-cell death in the course of the disease, T2D. Approximately 90% of pancreatic tissue samples taken post-mortem from T2D patients contain islet amyloid primarily composed of hIAPP . The extAlthough a link between hIAPP aggregation and pancreatic β-cell death is well established, precisely which type of hIAPP aggregate contributes to pancreatic β-cell death and insulin dependence remains undetermined. Using mostly in vitro studies, researchers have presented evidence for toxicity of multiple types of hIAPP aggregates. Early studies suggest that amyloid fibrils are the primary cytotoxic species because preparations that contain fibrillar hIAPP were more cytotoxic than soluble preparations of the protein . Using cIn closer agreement with earlier studies, we find that hIAPP preparations that contain fibrils are cytotoxic to a rat pancreatic β-cell line, thus motivating us to determine the structure of the spine of hIAPP fibrils. If fibrils are a bona fide type of toxic aggregate in vivo, then determining the atomic structure of the spine of hIAPP fibrils is a logical approach for advancing our understanding of disease-relevant targets . FurtherTo compare the cytotoxic effects of oligomeric and fibrillar hIAPP, we generated hIAPP preparations that contained either amyloid oligomers or fibrils. We did this by aging the same concentration of hIAPP for 0 and 24 h time periods. Aging hIAPP for 24 h yielded amyloid fibrils and no detectable oligomers as assessed by Thioflavin-T (ThT) binding, negative-stain transmission electron microscopy (TEM), and a dot blot assay using the fibrillar oligomer-sensitive antibody, LOC . Aging hWe observe that hIAPP preparations that contain fibrils are significantly more cytotoxic to rat pancreatic β-cells than hIAPP preparations that contain oligomers but no detectable fibrils . We assaAlthough we do not detect oligomers in the 24 h sample, we cannot rule out the possibility that it may contain some undetectable population of slowly forming, yet highly toxic oligomers that associate with fibrils. Despite this possibility, we chose to focus on studying fibrillar structures of hIAPP.Given that hIAPP fibrils are cytotoxic, we sought to identify the residues that compose their amyloid spine. We identified residues 15–29 as the amyloid spine based on several lines of evidence and previous work by others . First, For these reasons, we chose to focus on two overlapping 11-residue segments within this region of the sequence: residues 19–29 and residues 15–25. We chose to study the WT and early onset S20G mutation segments . All fouTo determine the structure of segment 19–29 S20G, we used Micro-Electron Diffraction (MicroED). MicroED employs a standard cryo electron microscope (cryoEM) in diffraction mode for data collection from 3D crystals only a few hundred nanometers thick  Shi et . Such th2 of surface area per strand, which equates to 24 Å2 per residue. This interface is one of the largest and most complementary of any structurally determined steric-zipper interface  that creates a kink in the peptide backbone. To investigate this hypothesis, we generated a model of 19–29 WT consisting of a mated pair of ten-stranded sheets. The model was identical to the 19–29 S20G atomic structure with the exception that we adjusted the backbone torsion angles of Ser20 to comply with the allowed regions of the Ramachandran plot for a non-glycine residue. We compared the energies of the WT and S20G structures after minimization with FoldIt . The dry2 per residue) compared to the average steric-zipper (20.1 Å2 per residue). Hence, there is no dry interface between adjacent sheets in the crystal, and the structure seems labile compared to that of 19–29 S20G.The atomic structure of segment 15–25 WT, also determined using MicroED , shows aConsistent with our observation of unmated β-sheets in the atomic structure, we observe that 15–25 WT fibrils are relatively weak and reversible compared to 19–29 S20G fibrils, which possess a canonical pathogenic amyloid fibril architecture. Turbidity readings followed by negative-stain EM reveal that 15–25 WT fibrils completely disaggregate in the presence of heat and 1% SDS, but 19–29 S20G fibrils remain intact in up to 2% SDS .Similar to 19–29 S20G, the 15–25 WT atomic structure reveals curved β-sheets. The sheets possess one of the highest RMSD’s of sheets from planarity for any hIAPP protein segment structure determined to date .X-ray fiber diffraction and radial profile analysis of 15–25 WT and 15–25 S20G fibrils indicate they form structures similar to each other . Taken tNext we investigated the cytotoxic effects of the spine segments in order to determine if any of them were similarly cytotoxic to full-length hIAPP preparations. Although the cytotoxic mechanism of hIAPP is not fully understood, several reports show hIAPP induces mitochondrial dysfunction, alters cell metabolism, and initiates activation of pro-apoptotic machinery . Based oUsing MTT dye reduction, we observe that the labile 15–25 fibrils are not cytotoxic to HEK293 cells , whereasBased on our examination of the insoluble and soluble fractions of the cytotoxic 19–29 S20G sample, we determine that the cytotoxicity of 19–29 S20G mainly resides in its fibrillar form. We tested the cytotoxicity of the total, insoluble and soluble fractions of the 19–29 S20G sample to HEK293 cells using MTT dye reduction. We observe that the insoluble fraction, which contains amyloid fibrils, is similarly cytotoxic to the total , just asFurther evidence that 19–29 S20G may form the toxic spine of full-length hIAPP comes from our observation that (−)-epigallocatechin gallate (EGCG), a flavanol known to mitigate full-length hIAPP cytotoxicity by preventing hIAPP from forming fibrils , also miNext we tested whether the spine segments activate pro-apoptotic machinery using a FRET-based biosensor assay for monitoring caspase-3 activity in real-time . In thisUsing this system, we observe that segment 19–29 S20G elicits the most caspase-dependent cytotoxicity of the spine segments and segments 15–25 are not cytotoxic . SegmentGiven that the spine segments seed full-length hIAPP fibril formation and that 19–29 S20G and 15–25 WT fibrils elicit different cytotoxic effects, we investigated whether seeding with either of the spine segments alters hIAPP cytotoxicity. To do this, we prepared seeded hIAPP at 10 μM with 10% monomer equivalent of pre-formed seeds, the same conditions used in the ThT assay in Using MTT dye reduction, we observe that hIAPP seeded with non-toxic 15–25 WT fibrils is less cytotoxic than hIAPP alone, but hIAPP seeded with stable, toxic 19–29 S20G fibrils is similarly cytotoxic to hIAPP alone . LikewisThere are two possible explanations for the reduced cytotoxicity of the seeded 15–25 WT sample. First, the 15–25 WT seeds may seed a non-toxic species of full-length hIAPP, or second, the 15–25 WT seeds may interact with full-length hIAPP in some way that reduces its cytotoxicity.X-ray fiber diffraction and radial profile analysis of the hIAPP fibrils used in the cytotoxicity assay reveal that fibrils formed by seeding with stable, toxic 19–29 S20G fibrils have a slightly tighter packing than fibrils formed by seeding with labile, non-toxic fibrils. hIAPP fibrils formed by seeding with stable, toxic 19–29 S20G fibrils exhibit reflections indicative of shorter equatorial Bragg spacings than hIAPP seeded with labile, non-toxic 15–25 WT fibrils (9.0 Å versus 10.0 Å) . The tigIn 1901, when Dr. Eugene Opie first observed islet amyloid in post-mortem pancreata of T2D patients, he proposed a link between the islet amyloid and T2D . Over a These findings motivated us to determine the structure of the spine of hIAPP fibrils and elucidate structural features important for hIAPP cytotoxicity. To improve our likelihood of crystallization and structure determination, we selected four protein segments that span the spine. We discovered that segment 19–29 S20G forms a pair of β-sheets mated at a dry interface, a structure that shares key features with full-length hIAPP fibrils as described in the following paragraph. What’s more, the fibrillar form of 19–29 S20G is cytotoxic. In contrast, segment 15–25 WT forms an unusual arrangement of single, out-of-register β-sheets that are not cytotoxic. The divergence in structure and cytotoxicity of segments 19–29 S20G and 15–25 WT suggests that strong, stable intermolecular interactions are important features of cytotoxic amyloid proteins.The experiments of this study show that the 19–29 S20G atomic structure recapitulates many of the structural features and cytotoxic properties of hIAPP. First, preparations of 19–29 S20G that contain fibrils are cytotoxic, as is the case for full-length hIAPP. Second, X-ray fiber diffraction calculated from the dry interface of the 19–29 S20G atomic structure shares key features with fiber diffraction collected from full-length hIAPP fibrils. Third, segment 19–29 S20G elicits cytotoxicity by altering cell metabolism and activating pro-apoptotic machinery, mechanisms by which full-length hIAPP fibrils are thought to contribute to pancreatic β-cell death during T2D. Fourth, the early onset S20G mutation confers greater cytotoxicity within segment 19–29 and within full-length hIAPP. Last, EGCG, a flavanol that mitigates full-length hIAPP fibril formation and cytotoxicity, likewise mitigates 19–29 S20G fibril formation and cytotoxicity. These results, taken together with the canonical pathogenic amyloid fibril architecture of segment 19–29 S20G, suggest it represents the toxic amyloid spine of hIAPP.Our studies begin to provide a framework for understanding which hIAPP fibril polymorphs may contribute to pancreatic β-cell death during T2D. Previous structural studies of hIAPP protein segments  and fullBoth atomic structures presented here reveal a new and potentially important observation: curved β-sheets. In the dry interface of the 19–29 S20G atomic structure, the curved β-sheets accommodate the tightly packed hydrophobic core, which includes a bulky phenylalanine, while maintaining high shape complementarity and large buried surface area. Paradoxically, in the 15–25 WT atomic structure, the curved β-sheets appear to have an opposite effect: the curved β-sheets appear to prevent adjacent sheets from associating to form a canonical pathogenic amyloid fibril architecture. In both atomic structures, the effect of the curved β-sheets is dictated by the registry of adjacent β-sheets .The 15–25 WT atomic structure joins the recently discovered class of out-of-register protein segment structures, which exert disparate cytotoxic effects. Here, we show that 15–25 WT is not cytotoxic but in contrast, the out-of-register protein segment KDWSFY from β2-microglobulin elicits mild cytotoxicity . One notAlthough the 19–29 WT fibrils prepared in this study appear morphologically similar to 19–29 S20G fibrils, the 19–29 WT fibrils are likely polymorphic and may contain some fraction of fibrils that are structurally similar to non-toxic 15–25 WT fibrils. Previous structural studies of segment 20–29 WT fibrils show that it forms an array of polymorphs, some of which are similar to the 15–25 WT atomic structure . StructuThese findings, expedited by MicroED, may inform our understanding of hIAPP fibril structures that contribute to pancreatic β-cell death in Type-II Diabetes patients. Going forward, we can use our toxic amyloid spine model as a template for structure-based design in the effort to develop much needed therapeutics that protect against pancreatic β-cell death and disease progression . In addi2 wild-type and mouse IAPP(1–37)NH2 wild-type were synthesized by Innopep  and CS Bio  and purified to greater than 98% purity. Human and mouse IAPP were prepared by dissolving the lyophilized proteins at 0.25–1 mM in 100% HFIP and leaving them to dissolve for several hours to ensure complete solubility. Next, the HFIP was removed with a CentriVap Concentrator . After removal of the HFIP, the peptides were dissolved at 1 mM, 5 mM, or 10 mM in 100% DMSO. The DMSO peptide stocks were diluted 100-fold in filter-sterilized Dulbecco’s PBS . Samples were incubated at room temperature for the designated time periods.Human IAPP(1–37)-NHAll four spine segments were synthesized by GenScript  and purified to greater than 98% purity. Fibrils were formed by dissolving lyophilized peptide at 1 mM in PBS and 1% DMSO.15-FLVHSSNNFGA-25 (15–25 WT). 15–25 WT was dissolved at 20 mg/ml in ice-cold, nano-pure water and then spin-filtered. Crystals were grown using the hanging drop vapor diffusion method at 4°C in 0.35 M NaSCN and 35% MPD. Crystals grew within several hours and reached maximum size in a week. 3D crystals only a few hundred nanometers thick grew alongside microcrystals in the same drops.19-SGNNFGAILSS-29 (19–29 S20G). Microcrystals were grown using the hanging drop vapor diffusion method at 30°C in 0.2M acetate salts and 40% MPD. 3D crystals only a few hundred nanometers thick were grown in batch by dissolving lyophilized peptide at 1 mM in PBS and 1% DMSO without seeding. Crystals grew on the bench top at room temperature in several hours.The procedures for MicroED data collection and processing largely follow published procedures . Briefly−/Å2 per second being deposited onto our crystals.Crystals that appeared visually undistorted and that were 100–300 nm thick produced the best diffraction. Datasets from individual crystals were merged to improve completeness and redundancy. Each crystal dataset spanned a wedge of reciprocal space ranging from 40–80°. We used a selected area aperture of approximately 1 μm. The geometry detailed above equates to an electron dose rate of less than 0.01 ehttp://cryoem.janelia.org/downloads)  .We used XDS to index and integrate the diffraction images and XSCALE  for mergFor 19–29 S20G, data from six crystals were merged to assemble the dataset used for molecular replacement. Of note, the resolution was cut off at 1.9 Å to facilitate subsequent rounds of structure refinement.For, 15–25 WT, data from six crystals were merged to assemble the dataset used for molecular replacement. Of note, the diffraction pattern from the 15-25 WT crystals diffracted with MicroED reveal a pseudo two-fold symmetry. In line with this observation, we indexed and integrated the diffraction images with space group C2, but the datasets had relatively poor statistics compared to the P1 datasets and our attempts at refining molecular replacement solutions from the C2 datasets failed. 19–29 S20G. We determined the structure using molecular replacement. An idealized 7-residue poly-alanine strand led us to our atomic model. The solution was identified using Phaser . A datas15–25 WT. We determined the structure using molecular replacement. Dozens of search models were used, but an out-of-register β-strand model led us to our solution. The solution was identified using Phaser . SubsequCalculations of the area buried and shape complementarity (SC) were performed with AREAIMOL  and SC , respect30 μL of human and mouse IAPP preparations used in the cytotoxicity assays in AB_2307391; Cat. #111-035-144, Jackson ImmunoResearch, West Grove, PA) diluted 1:10,000 in PBS-T for 1 hr at RT. The membrane was washed three more times, and then the signal was developed with Clarity Western ECL Substrate  and documented with a CCD camera. Exposures ranging from 5 s to 5 min were collected, but the 5 min exposure was used in all figures.1 μL of each sample generated for cytotoxicity assays in Samples were spotted onto grids (holey or non-holey) and allowed to settle on the grid for 160 to 180 s. Remaining liquid was wicked off and grids were left to dry before analyzing. Sample grids were analyzed on the TF20 Electron Microscope . Images were collected at 3500 or 6000x magnification with an additional 1.4x post-column magnification and recorded using a TIETZ F415MP 16 megapixel CCD camera.Samples for negative-stain EM were spotted on non-holey carbon-coated grids andallowed to settle on the grid for 160 to 180 s. Remaining liquid was wicked off and then 2% uranyl acetate was applied to the grid. After 1 min, the uranyl acetate was wicked off. The grids were left to dry before analyzing on the T12 Electron Microscope (FEI). Images were collected at 3,200 or 15,000x magnification and recorded using a Gatan 2kX2k CCD camera.CVCL_2177; Cat. # CRL-2058, Manassas, VA). Cells were cultured in RPMI media  plus 10% heat-inactivated fetal bovine serum. Cells were cultured at 37°C in a 5% CO2 incubator. They tested negative for mycoplasma using a MycoAlert PLUS Detection Kit  and they were authenticated using Cytochrome C Oxidase 1 (COX1) gene analysis by Laragen .Rin5F cells were purchased from ATCC (RRID:CVCL_6974). Cells were cultured in DMEM media  plus 10% heat-inactivated fetal bovine serum and 1% pen-strep (Life Technologies). Cells were cultured at 37°C in a 5% CO2 incubator. They tested negative for mycoplasma using a MycoAlert PLUS Detection Kit and they were authenticated using STR profiling (Laragen).HEK293 c18 cells (hereon referred to as HEK293) were a gift from Carol Eng in the laboratory of Arnold J. Berk at UCLA, but they were originally purchased from ATCC . Cells were cultured in RPMI 1640 with 11 mM glucose (Sigma) with 10% FBS, and 1% pen-strep. Cells were cultured at 37°C in a 5% CO2 incubator. They tested negative for mycoplasma using a PCR-based method and they were authenticated using mRNA analysis.CHO cells were purchased from ATCC . The samples were concentrated 10x in water and applied between two capillary ends and then the samples were left to dry overnight. Dried fibrils of spine segments and full-length hIAPP in Radial profiles were calculated using a program written in-house. The program calculates the average intensity as a function of distance from the beam center.Thioflavin-T (ThT) assays were performed in black 96-well plates  sealed with UV optical tape. hIAPP and mIAPP were dissolved at 1 mM in 100% HFIP. The peptides were then diluted 100-fold in 20 mM sodium acetate pH 6.5 and 10 μM ThT. Unsonicated fibril seeds were added at 1 μM monomer equivalent concentration (10% v/v). ThT fluorescence was recorded with excitation and emission of 444 nm and 482 nm, respectively, using a SpectraMax M5 . Experiments were performed in quadruplicate and readings were recorded every 3 min.SCR_003788)   and compHEK293 cells and Rin5F cells were plated at 10,000 and 27,000 cells per well in 90 μL, respectively, in 96-well plates . Cells were allowed to adhere to the plate for 20–24 hr.For the assay in For the assays in For all assays, 10 μL of sample was added to cells. By doing this, samples were diluted 1/10 from in vitro stocks. Experiments were done in triplicate.The appropriate statistical test for significance was determined by assessing whether (1) The sample sets had a Gaussian distribution using a D’Agostino-Pearson omnibus normality test and (2) The sample sets had equal variance using a Bartlett’s test or F test. For samples with Gaussian distributions and equal variances, we employed an unpaired t-test with equal standard deviations. For samples with Gaussian distributions, but unequal variances, we employed an unpaired t-test with Welch’s correction. For samples with non-Gaussian distributions and unequal variances, we employed a Mann-Whitney U-test.After a 24 hr incubation of samples with cells, 20 μL of Thiazolyl Blue Tetrazolium Bromide MTT dye  was added to each well and incubated for 3.5 h at 37°C under sterile conditions. The MTT dye stock is 5 mg/mL in Dulbecco’s PBS. Next, the plate was removed from the incubator and 100 μL of MTT stop solution  was added to each well. We ensured the MTT crystals were fully dissolved by placing the plates on an orbital shaker (slow speed) for about an hour prior to taking measurements. Alternatively, the MTT assay was stopped by carefully aspirating off the culture media and adding 100 μL of 100% DMSO to each well. Absorbance was measured at 570 nm using a SpectraMax M5. A background reading was recorded at 700 nm and subsequently subtracted from the 570 nm value.Cells treated with vehicle alone (PBS + 0.1% DMSO) were designated at 100% viable, and cell viability of all other treatments was calculated accordingly.For the MTT reduction assay in We used the caspase-3/7 GLO assay  to detect caspase-3/7 activation. For this assay, Rin5F cells were plated as previously described in white-walled 96-well plates . After the designated aging period of each hIAPP preparation, 10 μL of sample was added to cells and thus diluted 1/10 from in vitro stocks. Experiments were performed in triplicate. Samples were incubated with cells for 24 h. Next, cell culture media, caspase-3/7 reagent, and the cells were brought to room temperature. All media was aspirated from wells and then replaced with 25 μL of media and 25 μL of caspase-3/7 reagent and mixed thoroughly. The plate was incubated at room temperature for 30 min and then luminescence was measured using a SpectraMax M5. Experimental points were normalized to vehicle-treated cells, which were designated as 100%. Cells treated with 2 μM staurosporine were used as a positive control to ensure the assay kit worked correctly.CHO cells were stably transfected with a vector producing EYFP and ECFP connected via a short linker containing the Asp-Glu-Val-Asp (DEVD) sequence targeted by activated caspase-3. The short linker allows fluorescence energy transfer (FRET) to occur between the two fluorophores. During apoptosis activated caspase-3 cleaves the linker resulting in a loss of FRET measured as a reduced 540 nm/480 nm emission ratio.2, 1.2 mM MgSO4, 0.5 mM KH2PO4, pH 7.4) supplemented with 20 mM HEPES and 2 mM glucose (KRHG).Cells were plated at 25,000 cells per well in black 96-well optical bottom plates  and the assay was performed in Krebs-Ringer   were mixed with sonicated, preformed fibrils (seeds) made of the same peptide (corresponding to 166 nM of monomers) and immediately added to the plated cells. FRET was monitored in real-time by measuring emission at 480 nm and 540 nm with 440 nm excitation in a FLUOstar Omega microplate reader (BMG Labtech) over 24 hr at 37°C.Fibrils of 15–25 WT and 19–29 S20G at monomer equivalent concentrations were allowed to form for one week to ensure complete fibril formation. The samples were homogenized with vortexing, and then aliquoted to 0.5 mL tubes with equal volumes. Each fibril sample was treated with water or increasing amounts of SDS, and then heated at 55°C for 20 min. Next, an aliquot of each sample was transferred to a 384-well plate and turbidity was measured by recording absorbance at 340 nm. Each fibril sample was spotted onto a grid for negative-stain EM to analyze fibril abundance. The experiment was repeated twice, but the results of 1 experiment are shown in  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor, Nir Ben-Tal, and Michael Marletta as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Atomic Structures of Fibrillar Segments of Islet Amyloid Polypeptide Suggest Tightly Mated β-Sheets Drive Cytotoxicity" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:eLife.The manuscript addresses the important and controversial topic of human islet amyloid polypeptide (hIAPP) toxicity via a mostly structural study. The work is technically excellent, the use of Cryo electron microscopy-based MicroED is highly suitable for the scientific quest and the addition of the biological data is very important. The main conclusion is that the fibrillar form of hIAPP segment 19-29S20G is toxic, but its oligomeric form is not. In addition, in- or out-of registration/hydrophobicity of the four IAPP segments of interest in fibrillation is insufficient to account for the observed differential toxicity. This study concludes that 19-29S20G could be used as a model system for examining hIAPP aggregation and toxicity. The work is elegant and it provides important insights on the cytotoxicity of the polypeptide that is associated with Type II diabetes. The conclusions are consistent with the experimental results and the Discussion is very good. On the other hand, it is somewhat surprising that this manuscript heavily emphasizes on cytotoxicity – a not so very original point of this study, while the more novel structural observations are only mentioned in passing. If revised, it could be suitable for Essential revisions:1) The authors make it appear as if fibrils (and not oligomers or other intermediates) are generally considered as the major cytotoxic species of IAPP. However, there is very abundant literature evidence that oligomers and not fibrils are the major cytotoxic species, and the current study also does not explicitly analyze oligomer samples. The current description might be motivated by bad experiences with journal editors or referees who frequently dismiss all work on fibrils as being irrelevant and notoriously believe the pathology of any protein misfolding disease to solely depend on oligomers. This concept, while being too simple minded, is wide spread and causes numerous problems when attempting to publish solid scientific work. Yet, I also think that it is problematic to neglect the considerable body of published evidence in favor of the toxicity of IAPP oligomers, although there are also studies reporting the in vitro toxicity of fibrils, at least in certain set ups. Therefore, the following sentences should be changed:"Researchers have accumulated substantial evidence that supports a causative link between hIAPP aggregation and islet cell death leading to T2D" and "Consistent with this causation, mouse IAPP does not aggregate and mice do not get T2D. However, mice can be induced to develop islet amyloid and T2D when they are engineered to express human IAPP and fed a high fat diet . Perhaps the strongest support for a causative link is the mutation in hIAPP, hIAPP-S20G; segments containing this mutation aggregate more quickly and lead to early onset T2D in families who carry this lesion" suggest that IAPP toxicity causes T2D (type 2 diabetes). We think that no medical scientist would agree to this notion. Instead, IAPP aggregation is rather thought to constitute a secondary complication of T2D that actually develops for other reasons. The above sentences should be removed or changed such that they no longer imply that IAPP aggregation causes T2D."The causative link between hIAPP amyloid fibril formation and islet cell death is widely accepted, but researchers have debated whether soluble, pre-fibrillar oligomers or amyloid fibrils are the primary cytotoxic species that drive islet cell death" and "researchers suggested that amyloid fibrils are the primary cytotoxic species because hIAPP is only cytotoxic in its fibrillar form", "if hIAPP fibrils are the primary cytotoxic species, then determining the atomic structure of the spine of hIAPP fibrils is a logical approach for advancing our understanding of disease-relevant targets", and "we focused on studying fibrillar structures of hIAPP as the more relevant to cytotoxicity". These statements turn the currently dominating belief in the field rather upside down. Pre-fibrillar oligomers and other intermediates (and not fibrils) are nowadays considered to be the major toxic species of IAPP, although this evidence is largely based on in vitro studies and the in vivo pathogenic agents remain to be established. This view can be found, for example, in the very influential review from G. Westermark , one of the coauthors of the current study, where it says that "the mature amyloid fibril is presumed to be relatively inert and to have no significant cell toxicity. Rather, smaller oligomeric intermediates formed during fibrillogenesis are thought to be cytotoxic". The above statements should be amended.The following statement was used to support that residues 15-29 are important for toxicity: "previous work by our laboratory has shown that Phe15 is required for stabilizing an on-pathway α-helical dimer and that mutating this residue can delay aggregation". However, it solely describes aggregation and not toxicity. Please amend."The cytotoxicity of hIAPP resides mainly in its fibrillar form" and "here, we offer support for mature amyloid fibrils as a major type of aggregate responsible for islet cell death by showing that fibrillar hIAPP is significantly more cytotoxic to cultured pancreatic β-cells than pre-fibrillar forms". These statements are questionable as the presently reported cytotoxicity measurements did not explicitly test oligomer samples.The origin of divergence in the literature regarding the identity of the toxic entity perhaps has to do with the rapid fibrillation rate of hIAPP compared with, say, amyloid-β, where isolation of hIAPP monomers, oligomers, protofibrils and mature fibrils is nontrivial, as the authors also acknowledged. Since toxicity is the biological endpoint of a chemical , physical  or biological  trigger, it seems to make more sense that it is the combined contributions of these aspects, different for hIAPP fibrils and oligomers in each aspect, induce the toxicity of hIAPP.2) If indeed the fibrillar form of the polypeptide is the toxic element, could antibodies raised against this form be a treatment for Type II diabetes? Unlike the case of amyloid-β vaccination, the risk of meningoencephalitis is not a concern.3) What could be the cause that out of registration amyloid proteins behave so differently, as acknowledged by the authors in the Discussion (fifth paragraph)? Does it suggest that the structural properties of protein monomers/amyloids may not be the only source to explain toxicity?4) It might be beneficial to compare the overall hydrophobicity and charge of the four segments to decipher their differential toxicity as reported in this paper.5) All "cytotoxicity" data in 6) 7) 8) What could/should be emphasized much more clearly  are the following issues:A) The current cross-β sheets are concave or convex when you look down the spine axis. This curvature is a major difference to Madine et al., 2008, and probably also most previous steric zippers consisting of short chains. What does this curvature mean or what enforces it?B) Associated with this observation, C) Most steric zippers have the strands oriented perpendicular to the sheet long axis. This is not the case in D) How does the current packing of the zippers relate to the ones seen in earlier zipper described for IAPP ? Do the small zippers show the same interactions as the large ones? If not, what does it mean for the specificity of the side-chain interactions in the fibril? Essential revisions:1) The authors make it appear as if fibrils (and not oligomers or other intermediates) are generally considered as the major cytotoxic species of IAPP. However, there is very abundant literature evidence that oligomers and not fibrils are the major cytotoxic species, and the current study also does not explicitly analyze oligomer samples. The current description might be motivated by bad experiences with journal editors or referees who frequently dismiss all work on fibrils as being irrelevant and notoriously believe the pathology of any protein misfolding disease to solely depend on oligomers. This concept, while being too simple minded, is wide spread and causes numerous problems when attempting to publish solid scientific work. Yet, I also think that it is problematic to neglect the considerable body of published evidence in favor of the toxicity of IAPP oligomers, although there are also studies reporting the in vitro toxicity of fibrils, at least in certain set ups. Therefore, the following sentences should be changed:We have amended the manuscript to note that several current studies of hIAPP have implicated oligomers as the toxic agents . Thank you for suggesting this point."Researchers have accumulated substantial evidence that supports a causative link between hIAPP aggregation and islet cell death leading to T2D" and "Consistent with this causation, mouse IAPP does not aggregate and mice do not get T2D. However, mice can be induced to develop islet amyloid and T2D when they are engineered to express human IAPP and fed a high fat diet . Perhaps the strongest support for a causative link is the mutation in hIAPP, hIAPP-S20G; segments containing this mutation aggregate more quickly and lead to early onset T2D in families who carry this lesion" suggest that IAPP toxicity causes T2D (type 2 diabetes). We think that no medical scientist would agree to this notion. Instead, IAPP aggregation is rather thought to constitute a secondary complication of T2D that actually develops for other reasons. The above sentences should be removed or changed such that they no longer imply that IAPP aggregation causes T2D.We have amended the manuscript to remove any assertion of causation. We do, however, point out the evidence showing a correlation of hIAPP aggregation to pancreatic β-cell death ."The causative link between hIAPP amyloid fibril formation and islet cell death is widely accepted, but researchers have debated whether soluble, pre-fibrillar oligomers or amyloid fibrils are the primary cytotoxic species that drive islet cell death".In the amended manuscript we have removed the term “causative”.And "researchers suggested that amyloid fibrils are the primary cytotoxic species because hIAPP is only cytotoxic in its fibrillar form", "if hIAPP fibrils are the primary cytotoxic species, then determining the atomic structure of the spine of hIAPP fibrils is a logical approach for advancing our understanding of disease-relevant targets", and "we focused on studying fibrillar structures of hIAPP as the more relevant to cytotoxicity".These sentences have been amended in the revised manuscript  are nowadays considered to be the major toxic species of IAPP, although this evidence is largely based on in vitro studies and the in vivo pathogenic agents remain to be established. This view can be found, for example, in the very influential review from G. Westermark , one of the coauthors of the current study, where it says that "the mature amyloid fibril is presumed to be relatively inert and to have no significant cell toxicity. Rather, smaller oligomeric intermediates formed during fibrillogenesis are thought to be cytotoxic". The above statements should be amended.Thank you for noting the important difference between in vitro and in vivo studies, which we now mention in the third paragraph of the Introduction. We do not, however, get into the mechanism of hIAPP action on cells; we restrict ourselves to reporting our observations of toxicity without proposing a mechanism. Our private hypothesis is that the mechanism may involve both mature fibrils and smaller species, perhaps short fibrils. That is, amyloid fibril deposition leads to distortion of the histological structure of the affected organ. Islet hormone release is dependent on the islet architecture and regulated insulin release requires cell-cell interaction via connexin-36. When hIAPP amyloid deposits extracellularly it results in separation of the β cells and loss of synchronized hormone release. Islet amyloid occurs perivascular and sometimes prominent deposits occur, possibly influencing diffusion of oxygen and nutrients. Whereas mature amyloid fibrils may exert no direct β cell cytotoxicity, amyloid fibrils somehow affect islet function, and the results of our paper may contribute to the ultimate understanding of the mechanism.Although we do not propose a mechanism, we report one experiment that may bear on it. With our FRET-based amyloid toxicity assay, we showed that the amyloid fibril formation-process induced toxicity . An earlThe following statement was used to support that residues 15-29 are important for toxicity: "previous work by our laboratory has shown that Phe15 is required for stabilizing an on-pathway α-helical dimer and that mutating this residue can delay aggregation". However, it solely describes aggregation and not toxicity. Please amend.We amended the wording to make our intended statement clearer. It now reads: “Third, previous work by our laboratory has shown that Phe15 may be part of the amyloid spine because it is required for stabilizing an on-pathway α-helical dimer and mutating this residue can delay fibril formation .”"The cytotoxicity of hIAPP resides mainly in its fibrillar form" and "here, we offer support for mature amyloid fibrils as a major type of aggregate responsible for islet cell death by showing that fibrillar hIAPP is significantly more cytotoxic to cultured pancreatic β-cells than pre-fibrillar forms". These statements are questionable as the presently reported cytotoxicity measurements did not explicitly test oligomer samples.In order to address this revision and essential revisions #5 and #6, we repeated the cytotoxicity studies shown in Using the LOC antibody, we detect fibrillar oligomers in the “hIAPP 0 h” preparation. Although the LOC antibody was raised against hIAPP fibrils, previous studies show that it binds to fibrillar oligomers of amyloid-β . Fibrillar oligomers are structurally distinct from pre-fibrillar oligomers, which are recognized by the A11 antibody.Using two metrics of cytotoxicity, we find that hIAPP preparations that contain these fibrillar oligomers are significantly less cytotoxic than hIAPP preparations that contain fibrils and no detectable oligomers. The full description of these results starts in the subsection “hIAPP preparations that contain fibrils are cytotoxic to cultured rat pancreatic β-cells”.The above statements were amended to reflect our updated cytotoxicity studies. They now read: “We observe that hIAPP preparations that contain fibrils are significantly more cytotoxic to rat pancreatic β-cells than hIAPP preparations that contain oligomers but no detectable fibrils ”; and “IThe origin of divergence in the literature regarding the identity of the toxic entity perhaps has to do with the rapid fibrillation rate of hIAPP compared with, say, amyloid-β, where isolation of hIAPP monomers, oligomers, protofibrils and mature fibrils is nontrivial, as the authors also acknowledged. Since toxicity is the biological endpoint of a chemical , physical  or biological  trigger, it seems to make more sense that it is the combined contributions of these aspects, different for hIAPP fibrils and oligomers in each aspect, induce the toxicity of hIAPP.Good point. But we do not have evidence for mechanism, so we prefer not to discuss this thoroughly. However, we acknowledge the potential disconnect between toxic aggregates identified in vitro and toxic aggregates present in vivo in the third paragraph of the Introduction and we briefly mention the possibility of multi-variable toxicity in the sixth paragraph of the Discussion.2) If indeed the fibrillar form of the polypeptide is the toxic element, could antibodies raised against this form be a treatment for Type II diabetes? Unlike the case of amyloid-β vaccination, the risk of meningoencephalitis is not a concern.Presumably, yes. We mention this in the Discussion of this revised submission (last paragraph).3) What could be the cause that out of registration amyloid proteins behave so differently, as acknowledged by the authors in the Discussion (fifth paragraph)? Does it suggest that the structural properties of protein monomers/amyloids may not be the only source to explain toxicity?One potential cause for the different cytotoxic effects of the 2 out-of-register protein segments is whether or not the out-of-register sheets are mated. Mildly cytotoxic protein segment KDWSFY from β-2 microglobulin has sheets mated by a dry interface, but non-toxic hIAPP 15-25 WT is composed of single sheets. The dry interface of the KDWSFY crystal structure results in higher solvation energy per strand compared to the 15-25 WT crystal structure . Given that cytotoxic structures like 19-29 S20G have high solvation energies per strand .4) It might be beneficial to compare the overall hydrophobicity and charge of the four segments to decipher their differential toxicity as reported in this paper.We thank the reviewers for making this suggestion. To investigate this and to compare the structures of shorter hIAPP protein segments previously determined, we added Spine segments 19-29 S20G and 15-25 WT possess similar charges  and only slightly different hydrophobicity values  compared to other hIAPP protein segments for which we have crystal structures. Furthermore, the overall charge and hydrophobicity values for cytotoxic full-length hIAPP, (2.1 at pH 7 and – 0.097) are not particularly similar to any of the charges or hydrophobicity values for the protein segments described here. It is difficult to associate 1 biophysical characteristic with cytotoxicity as we only have cytotoxicity data for the 4 protein segments described in this study. Nonetheless, since we observe that the hydrophobicity values for the protein segments described here are close to the average hydrophobicity value for all hIAPP protein segments ever characterized (average = 0.11), we presume overall hydrophobicity and charge of the segments do not fully explain their differential toxicity. It seems more likely that their differential cytotoxicity is better explained by their divergent structures.5) All "cytotoxicity" data in We acknowledge the potential issues of the MTT assay. Of note, in the JBC paper cited by the reviewers, the researchers discovered that only fragmented fibrils, not unfragmented fibrils like those used in our studies, produced false positive results in the MTT assay. Additionally, as the reviewers point out in essential revision #1, our studies described in the original By halving the concentration of IAPP that we used in our original studies, we were able to generate oligomer and fibril preparations of hIAPP. In the “hIAPP 0 h” preparation, we detect only fibrillar oligomers and in the “hIAPP 24 h” preparation, we detect only amyloid fibrils.We tested the cytotoxicity of the samples using the MTT assay and a luminescence-based caspase-3/7 detection assay in parallel. Active caspase-3 and caspase-7 proteases are markers of apoptosis . Both assays show that “hIAPP 24 h,” which contains fibrils and no detectable oligomers, is significantly more cytotoxic than “hIAPP 0 h,” which contains only oligomers. Furthermore, we find that the insoluble fraction of the cytotoxic “hIAPP 24 h” sample is more cytotoxic than the soluble fraction of the sample, providing more evidence for cytotoxicity of fibrils in our studies. We added the results of these studies to 6) As stated in our response to essential revisions #1 and #5, we repeated the cytotoxicity studies described in the original Additionally, we performed dot blot assays of the samples used in the cytotoxicity studies with 25 different conformational antibodies known to recognize soluble oligomers. Only one of these antibodies, LOC, gave a robust and consistent result across biological triplicates. We replaced the original dot blot results with the LOC dot blot results in 7) We agree with the reviewers’ interpretation of the result with hIAPP seeded with toxic 19-29 S20G fibrils. This result does not appear to show a templating of toxicity by a fragment onto full- length hIAPP. However, the result with hIAPP seeded with non-toxic 15-25 WT fibrils may be explained by (1) our interpretation of templating growth of fibrils of relatively low toxicity or (2) by the reviewers’ interpretation of seed interacting with hIAPP to reduce its toxicity. Both interpretations seem reasonable considering that 15-25 WT fibrils template full-length hIAPP fibril formation (8) What could/should be emphasized much more clearly  are the following issues:A) The current cross-β sheets are concave or convex when you look down the spine axis. This curvature is a major difference to Madine et al., 2008, and probably also most previous steric zippers consisting of short chains. What does this curvature mean or what enforces it?This subject is addressed more fully in the amended manuscript. The β-sheets in the crystal structures presented here are curved compared to shorter hIAPP protein segments previously determined. To quantify β-sheet deviation from planarity across all protein segment crystal structures, we calculated their standard deviation of atoms from a best-fit plane . Many ofOur private hypothesis is that kinking and curvature are two mechanisms by which β-sheets may increase the interactions with neighboring sheets. In other words, kinks and curvature impart plasticity to the otherwise strictly planar β-sheet so it may conform to the shape of an adjacent surface to produce tighter dry interfaces (as in the case of 19-29 S20G) or optimize crystal contacts (as in the case of 15-25 WT). Indeed, our modeling studies showed without the kink introduced by the S20G mutation, the 19-29 segment cannot form as tight of a dry interface (see last paragraph in the subsection “Segment 19-29 S20G forms pairs of β-sheets tightly mated by a dry interface”).We speculate that kinking and curvature are likely to be observed more frequently with increasingly longer segments, since longer segments are likely to have greater variation in side chain lengths and kinks and curves provide additional degrees of freedom for sheets of varying thickness to mate more tightly.Moreover, we think that kinked sheets are likely to be more common for in-register sheets than out-of-register sheets. We speculate that if the 15-25 WT segment could kink in addition to curving, the new shape would afford another chance that it may conform tightly with neighboring sheets. However, there is likely to be a high energy barrier to introducing a kink in the sheet formed by the 15-25 WT segment because the sheet lacks alignment of glycine residues, which assists with kink formation. Recall that kink formation requires a crease to propagate through the entire length of the sheet. Because the 15-25 WT structure is out of register, Gly24 residues do not align in a continuous line.B) Associated with this observation, Thank you for this suggestion. To investigate this question, we measured the standard deviation in sheet-to-sheet distance for all hIAPP protein segment structures determined to date. The variation of packing distance appears to accommodate the variation in side-chain sizes protruding from the sheet. This observation is noted in the revised text and it is also included in the newly added C) Most steric zippers have the strands oriented perpendicular to the sheet long axis. This is not the case in Indeed, most steric-zippers are formed of β-strands stacked perpendicular to the sheet long axis. We describe such zippers as “in-register.” A few years ago, our group discovered steric-zipper structures that are formed of β-strands that stack at an angle to the sheet long axis. We describe these zippers as “out-of-register.” Deviation of strands from the fibril perpendicular is a natural consequence of the registration shift implied by out-of-register structures. The 15-25 WT crystal structure presented in To date, only 4 out-of-register steric-zippers have been identified and the significance of the out- of-register stacking of β-strands is not yet well established. It is possible that the out-of-register stacking could infer tendencies for the sheet to roll into a cylinder, like cylindrin does . The 4 out-of-register steric-zippers are formed of protein segments derived from β-2 microglobulin , prion protein , and IAPP  suggesting out-of-register stacking is a shared feature of amyloid proteins.We expanded our description of out-of-register protein structures in the Results and Discussion sections.D) How does the current packing of the zippers relate to the ones seen in earlier zipper described for IAPP ? Do the small zippers show the same interactions as the large ones? If not, what does it mean for the specificity of the side-chain interactions in the fibril?Crystal structures of shorter hIAPP protein segments exhibit an array of sheet and side-chain interactions, which we term polymorphism. Even shorter protein segments that fully overlap with the 11-residue spine segments described in this work exhibit significant polymorphism. We list the steric-zipper classes and other structural features of each protein segment in the newly added Furthermore, full-length hIAPP fibril models derived from solid-state NMR studies  also suggest variable side-chain interactions in fibrils. Taken together with the 15 crystal structures of hIAPP protein segments, we surmise that there is some flexibility of side- chain interactions in the fibril.Nonetheless, the extensive size of the 19-29 S20G dry interface implies it is particularly stable. In comparison, the zippers in our earlier publications have fewer residues and hence, smaller interfaces.


Tractable experimental model that accounts for inter-tumor molecular heterogeneity is a key element of anti-cancer drug development. Hepatocellular carcinoma is known to exhibit highly heterogeneous molecular aberrations across the tumors, including somatic genetic and epigenetic alterations. Previous studies showed that molecular tumor subtypes determined by transcriptome, as a comprehensive functional readout, are reproducibly observed across global patient populations irrespective of geographic and etiological variations. Here we demonstrate that transcriptomic hepatocellular carcinoma subtypes, S1 and S2, determined by our previous transcriptome meta-analysis of multiple clinical hepatocellular carcinoma cohorts, are presented in a panel of hepatoma cell lines widely used by the research community. Interestingly, cell line that resembles gene expression pattern of S3 subtype, representing less aggressive tumors, was not identified in the panel. MYC pathway-activated S2-like cell lines showed higher sensitivity to a small molecule BET bromodomain inhibitor, (+)-JQ1, which has anti-MYC activity. These results support the use of hepatoma cell lines as models to evaluate molecular subtype-specific drug response, which is expected to lead to development of tailored, precision care of the patients with hepatocellular carcinoma. Potentially druggable somatic DNA mutations are generally rare , which makes patient enrollment in clinical trials more challenging.2 A recent study of patient-derived HCC cell lines demonstrated selective killing only in subclones that harbor the relatively rare somatic DNA aberrations such as FGF19 gene amplification.3Experimental models are key components of anti-cancer drug development and an urgent unmet need for hepatocellular carcinoma (HCC), which still lacks substantially effective medical therapy.5 The subtypes are associated with clinical characteristics such as serum α-fetoprotein (AFP) levels, and also with histological intra-tumor heterogeneity, suggesting their clinical relevance.5 It is empirically known that some of the transcriptomic subtypes are present in human hepatoma cell lines, with which subtype-specific in vitro and in vivo anti-cancer drug response has been demonstrated.8 In parallel, clinical features associated with the subtypes such as AFP (a marker of the S2 subtype) have been recognized as possible predictive biomarkers of therapeutic response to molecular targeted agents as shown in recent clinical trials.9 These findings collectively suggest that hepatoma cell lines can be used as tractable and clinically relevant experimental models of the HCC subtypes for assessment of molecular targeted anti-cancer agents. In this study, we have systematically evaluated the HCC subtypes and their association with therapeutic response in a panel of frequently and widely used hepatoma cell lines.Transcriptome is an alternative measure, which is assumed to be functional readouts of various types of molecular aberrations such as mutations and epigenetic changes. Our previous transcriptome meta-analysis revealed three subtypes, namely S1, S2 and S3, which are observed in larger fractions of patients/tumors  associated with specific molecular pathway dysregulation and patient prognosis.https://portals.broadinstitute.org/ccle/home). Pre-normalized genome-wide transcriptome data set of 374 human HCC tissues was downloaded from The Cancer Genome Atlas (TCGA) website (https://gdc.cancer.gov). In the CCLE hepatoma data set, transcriptome profile of PLC/PRF/5 cell line duplicated in an alternative name (Alexander) was excluded. Hepatoblastoma-derived cell lines, Hep G2 and its derivative C3A,10 and undifferentiated hepatoma cell lines, HLE and HLF, were included in the analysis given their wide use in the HCC research community. Transcriptomic subtyping of the cell lines was performed based on the HCC subtype maker gene signatures by using the Nearest Template Prediction algorithm as previously reported.11 Non-negative matrix factorization (NMF)-based consensus clustering was used to explore optimal number of classes in the global transcriptome data set after excluding less variable probes based on a coefficient of variation (CV) cutoff of 0.5.12Pre-normalized genome-wide transcriptome and DNA mutation data sets of 25 hepatoma cell lines  were dow−1 penicillin  and 100 μg ml−1 streptomycin (Gibco) at 37 °C in a 5% CO2 atmosphere.SNU-182, SNU-387, SNU-475, SNU-449, Hep G2, Hep 3B2.1-7, Huh-7 and THLE-5B cells  were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U ml5 Raw transcript count data were normalized by scaling with geometric mean of built-in normalization genes. HCC subtype determination was similarly performed by the Nearest Template Prediction algorithm.Total RNA was extracted from the cell lines using RNeasy kit , and 200 ng total RNA was subjected to the Elements HCC subtyping assay  as previously reported.A total of 3000–5000 cells were plated in each well in 96-well plates according to growth rate of each cell line, and when 50% confluence was reached ~24 h later, treatment with (+)-JQ1  at varying concentrations was started and continued for 48 h in triplicates.2 atmosphere for 4 h. The absorbance was then read at 490 nm. Averaged absorbance from empty wells was subtracted from all wells as background signal, and relative cell viability was calculated as a percentage of the adjusted absorbance compared to respective DMSO-treated control wells.Cell viability was assessed using an MTS Cell Proliferation Assay . A volume of 22 μl of the MTS assay solution was added to each well that contained 110 μl of media. The plates were then incubated at 37 °C in a 5% COP-value <0.05 was regarded as statistically significant. All analyses were performed using R statistical language (www.r-project.org).Proportion of categorical data was assessed by Fisher’s exact test. Two-tailed EPCAM and AFP, and S3 subtype enriched with more differentiated tumors and accumulation of CTNNB1 exon 3 mutations associated with induction of liver-specific Wnt pathway target, GLUL.13 It has been reported that hepatoma cell lines are classified into two groups by unsupervised clustering of global transcriptome profiles, which is correlated with AFP expression status.15 AFP-high and -low cell lines were empirically known to exhibit transcriptome profiles similar to S2 and S1 subtypes, respectively.4 Consistent with these observations, non-negative matrix factorization consensus clustering of the genome-wide transcriptome profiles of 25 hepatoma cell lines from the CCLE data set revealed that optimal classification is indeed achieved when two subclasses are assumed (n=374), median expression levels of each HCC subtype marker genes in each sample  depicted relative abundance of each subtype marker gene expression in human HCC tissues , S2 subtype with overexpression of stemness markers such as  assumed . In the  tissues .CTNNB1 exon 3 mutations prevalent in the S3 subtype, was observed only in one cell lines (SNU-398) with relatively low expression of the target gene GLUL  as seen in clinical tissues, and HIF1A was the most frequently affected gene by mutations (68%). Prevalence of these mutations was similar between the HCC subtypes for TP53 , HIF1A (P=0.38), TERT promoter (P=1.0) and rest of the genes recurrently affected in human HCC tissues (P=0.83).2In the hepatoma cell lines, the relative expression levels of the S1 and S2 subtype marker genes were maintained, whereas the S3 subtype marker genes were not expressed at the levels in tissues, suggesting that the hepatoma cell lines generally lack characteristic of the S3 subtype. In fact, the ene GLUL  and 2. T5 The HCC subtype determination based on the genome-wide transcriptome data  in an FDA-approved diagnostic assay platform (NanoString) for clinical application in our previous study.ome data  was veri5B cells . This re5B cells .in vitro and in vivo subtype-specific drug response by targeting fibroblast growth factor receptor pathway, one of the molecular hallmarks specifically activated in the S2 subtype.8 We sought to extend this examination to another pathway implicated in HCC subtypes. MYC pathway is known to be activated in the S2 subtype.4 Direct targeting of this pathway has been technically challenging, but BET bromodomain inhibition by a small molecule, (+)-JQ1 has been show to elicit anti-MYC pathway activity.18 To evaluate whether the subtype-dependent response to MYC pathway inhibition can be monitored in hepatoma cells, SNU-387 and SNU-182 (S1-like cells) as well as Huh-7 and Hep G2 (S2-like cells) were treated with (+)-JQ1. In the CCLE data set, expression of the target genes, BRD2, BRD3 or BRD4, at naive state was confirmed in the S2-like cell lines, which are already known to exhibit MYC pathway activation -JQ1 as indicated by lower half maximal inhibitory concentration (IC50) values  values , furtherin vitro models of human HCC molecular subtypes. Alternative strategies such as patient-derived xenograft (PDX) could enable personalized molecular evaluation for each patient.19 However, the low success rate and long time required to establish viable lines limit their flexible use. In contrast, despite the well-recognized limitations such as lacked tissue architecture and crosstalk with other cell types, cancer cell lines remain one of the major experimental models for anti-cancer drug discovery and assessment as a tractable system easily amenable to high-throughput and high-content molecular analysis.21This study provides comprehensive reference information for a panel of widely used and commercially available hepatoma cell lines as 6 S2-like cells were more susceptible to epidermal growth factor receptor (EGFR) and insulin-like growth factor 1 receptor (IGF1R) inhibition.23 BGJ398 (pan-fibroblast growth factor receptor inhibitor) elicited in vivo anti-tumor effect in xenograft of the S2-like cells.8 Epitope-optimized genetic vaccines targeting AFP (S2 marker)-induced specific CD8 T-cell-mediated killing of HCC cells in mice.24 Galunisertib, which targets the TGFβ pathway, a hallmark of the S1 subtype, is currently under evaluation in a phase 2 trial .25 Tivantinib (MET inhibitor),26 ramucirumab 9 and GC33 28 were tested in clinical trials and could be more effective in the S2 subtype.Modeling of specific disease contexts such as clinical/molecular tumor subtypes is generally attempted by genetic engineering in experimental systems. However, it is practically infeasible to reconstitute the full spectrum of complex genetic and epigenetic molecular aberrations generally observed in non-hereditary solid cancers. Our study highlights an advantage of using existing hepatoma cell lines that inherently exhibit and recapitulate naturally occurring transcriptomic dysregulation observed in two of the three consensus human HCC molecular subtypes, which represent more advanced and aggressive tumors and therefore should be prioritized for therapeutic development. In fact, multiple preclinical studies and clinical trials support this concept. Dasatinib (Src/Abl inhibitor) was more effective in the S1-like cells.In our analysis of 25 hepatoma cell lines and one immortalized hepatocyte line, we could not find any cell line with the features of the S3 subtype. This may indicate that HCC cells in S3 tumors, clinically more differentiated and associated with better prognosis, are not capable of survival and proliferation in culture without support from the physiological liver tissue architecture. It is important to note that there is certain diversity in the characteristic molecular features for each subtype between the cell lines. Therefore, presence or absence of specific features , which are potentially relevant to mechanism of action for agents to be tested, must be confirmed before picking cell lines for evaluation. In addition, differences in the sources and/or passages could alter the status of transcriptomic subtypes and other genetic aberrations, which ideally should be verified prior to the assessment. In summary, noting these limitations, hepatoma cell lines with designated HCC subtypes represent an invaluable resource as experimental models that retain the intra/inter-tumor/patient heterogeneity for anti-cancer drug assessment and development.


MYC gene amplification were positive for MYC by IHC. Four cell lines with high MYC expression and four with low MYC expression were used in further analysis. A spliceosome inhibitor, pladienolide B, showed high efficacy (IC50 < 12nM) in all eight cell lines tested, irrespective of the MYC IHC or MYC gene amplification status. We observed that the four cell lines with higher sensitivity to the spliceosome inhibitor were established from patients with prior chemotherapy. Therefore we chronically treated H1048 cells, that were established from a treatment-naïve patient, with cisplatin for 4 weeks, and found that H1048-cisplatin treated cells became more sensitive to pladienolide B. In conclusion, our in vitro results indicate that spliceosome inhibitors would be promising molecular target drugs in SCLC irrespective of the MYC status, especially in the second-line settings after an effective front-line chemotherapy.Small cell lung cancer (SCLC) is a highly aggressive malignancy with few therapeutic advances in the treatment in recent decades. Based on a recent study that identified the spliceosome as a therapeutic vulnerability in MYC-driven breast cancers, we evaluated the efficacy of a spliceosome inhibitor in SCLC cell lines and analyzed the correlation with MYC status. Among 23 SCLC cell lines examined, eight showed high MYC protein expression (> 80% positive cells) by immunohistochemistry (IHC), while 10 cell lines demonstrated no staining for MYC. The remaining five cell lines showed weak staining (< 40% positive cells). All four cell lines that were previously demonstrated to have  Small cell lung cancer (SCLC) accounts for about 15–20% of lung cancer diagnoses, and is one of the most aggressive type of cancer with high mortality. Despite a recent development of molecular targeted therapy in non-small cell lung cancers [MYC family genes [MYC family genes, MYC , MYCL, or MYCN in a mutually exclusive manner, was recognized in SCLC two decades ago [The widely known genetic alterations in SCLC are inactivating mutation/deletion of TP53 and RB1 and amplification of ly genes –5. Ampliades ago . Howeverades ago .MYC gene amplification or MYC overexpression.A recent study found that the spliceosome, a dynamic macromolecular ribonucleoprotein (RNP) complex that catalyses the splicing of nuclear pre-mRNA into mRNA, is a therapeutic vulnerability in breast cancer models driven by MYC, due to MYC-induced elevation of mRNA synthesis . To eval2 in a cell culture incubator. A spliceosome inhibitor, Pladienolide B, was purchased from Santa Cruz Biotechnology, Inc. . Cisplatin was purchased from Selleck Chemicals .A total of 23 human SCLC cell lines were used in this study. All cell lines were in our archive or a kind gift from our collaborators. The short tandem repeat profiles of all cell lines used have been confirmed. All cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 1x penicillin / streptomycin solution . Cells were grown at 37°C with 5% COFormalin-fixed paraffin-embedded (FFPE) cell blocks were prepared to make a cell line tissue microarray (TMA). The TMA was sectioned at a thickness of 4 μm, and mounted on charged glass slides. MYC IHC staining was performed on a Ventana Discovery Ultra autostainer employing a c-MYC rabbit monoclonal antibody . MYC staining was assessed by the study pathologist (H.Y.) using the H-score assessment which combines staining intensity (0–3) and the percentage of positive cells (0–100%).3) were plated into each well of 96-well flat-bottomed plates and cultured for 24 hours. Pladienolide B, cisplatin, or dimethyl sulfoxide (DMSO) was added to the indicated drug concentration, and cells were incubated for an additional 72 hours. A colorimetric activity assay was performed by addition of the PrestoBlue reagent to each well and the plates incubated at 37°C followed by fluorescence detection (560nm Ex / 590nm Em) using a Biotek Synergy II plate reader. Percent growth was calculated relative to DMSO-treated controls. Statistical differences between growth curves was assessed using the ‘compareGrowthCurves’ function of the statmod software package (http://bioinf.wehi.edu.au/software/compareCurves).Cell proliferation was measured using the PrestoBlue Cell Viability Reagent , according to the manufacturer’s instructions. Briefly, tumor cells  –11 or thMYC gene amplification [Representative MYC IHC staining patterns are shown in fication , thirteeMYC gene amplification status, showed high sensitivity to Pladienolide B with an IC50 value of less than 12 nM. There was no statistical difference between the high MYC group and negative/weak MYC group (p = 0.32).We then compared the growth inhibitory effect of a spliceosome inhibitor, Pladienolide B, in the high MYC group  and negative/weak MYC group . As shown in Because many of the SCLC cell lines were established from patients who had received chemotherapy, we divided these 8 cells lines into chemotherapy-naïve cell lines and those with prior chemotherapy based on the information written in the original papers ,13,14. WTherefore we chronically treated two cell lines (SW1271 and H1048 cells), that were established from treatment-naïve patients, with 5 uM of cisplatin for four weeks. The two cell lines were choses as they grow adherent (suspension cells are difficult to select viable cells during drug treatment). H1048 parental cells were highly sensitive to cisplatin, while SW1271 cells were not . AlthougDeregulation of MYC has long been recognized as playing a role in human malignancies, including lung, breast, colon, and prostate cancers, Burkitt’s lymphoma and other hematological malignancies . HoweverAmong these candidates, we focused on the spliceosome inhibitor based on the correlation of spliceosome inhibition and MYC activation as reported in breast cancer , lymphomMYC gene amplified cases from the analysis for SCLC patient specimens [50 < 12nM) irrespective of the MYC protein expression or MYC gene amplification status. We consider that this is likely due to high mRNA synthesis in SCLCs irrespective of MYC status. Of interest is a recent study that evaluated Omomyc, a MYC dominant negative, in a panel of SCLC cell lines. This study observed that Omomyc suppressed the growth of all cell lines tested irrespective of MYC status [We first examined MYC protein expression status in SCLC cell lines, because high MYC protein expression was also reportedly observed in non-pecimens . SubsequC status .Our results indicate that prior-chemotherapy affects the efficacy of the spliceosome inhibitor. The molecular background of this phenomenon is unclear, however we consider that cytotoxic chemotherapies may select cancer cells with higher mRNA synthesis. Long-term cisplatin treatment itself did not alter the efficacy of Pladienolide B in SW1271 cells with inherent cisplatin resistance, and this fact supports the above hypothesis.in vitro results indicate that spliceosome inhibitors would be promising molecular target therapy in SCLC irrespective of the MYC status, especially in the second-line settings after an effective front-line chemotherapy.In conclusion, our


Saccharomyces cerevisiae. Uniquely for [PSI+], the prion form of the Sup35 protein, there seem to be two activities, involving differing co-chaperones, by which Hsp104 affects the inheritance of [PSI+], the prion form of the Sup35 protein. Each pathway is also involved in protection against ageing, one through disaggregation of damaged proteins and the other through their retention in the mother cell during budding. Mutations in both Hsp104 and Sup35 affect prion inheritance by one or other of these pathways, as does manipulation of either Hsp104 enzyme activity or expression, in both vegetative (budding) divisions and in sporulation. Based on our recent finding (Ness et al. in Molec Microbiol 104:125–143, PSI+] cells in sporulation may be a marker for a role for Hsp104 in rejuvenation during sporulation.The AAA+ disaggregase Hsp104 is essential for the maintenance and inheritance of nearly all known prions of the yeast  E. coli, a protein, curli, which is excreted and helps in the formation of biofilms and in the yeast Saccharomyces cerevisiae, the [PSI+] prion, which is an aggregate of the polypeptide chain termination factor Sup35/eRF3  allows the prion to be diluted out during cell division due to the loss of fragmentation by this disaggregase  aggregates into lower molecular weight forms when Hsp104 is over-expressed. Rather what happens in many cell divisions is that the genetic units of prion inheritance, entities we call propagons  in a [PSI+] cell leads to the competitive inhibition of the wild type ATPase and the resulting prion loss mimics quantitatively the kinetics of GdnHCl-induced [PSI+] curing /Hsp70 (Ssa1) chaperones  and the SUP35 gene . Zygotes formed by crossing a PNM1.1 [psi−] strain with a ‘strong’ variant of [PSI+] 4 h after zygote formation, results in decoration of the propagons from the [PSI+] parent with Sup35p from the [psi−] cell  not HSP104 which leaves open the question whether the elimination of [PSI+] in this cross is due to an unusual sensitivity of the mutant protein to Hsp104 disaggregase. However, we find that dominant PNM1 mutants of Hsp104 totally lacking ATPase activity give results either quantitatively indistinguishable from those in Table The cross discussed above Table  involvedPSI+] in the PNM2G58R [psi−] × [PSI+] would not appear to be an amyloid disaggregation problem, but a propagon segregation problem. Nevertheless, in the absence of any perturbation of Hsp104 function by mutation or environment, all the empirical evidence is consistent with a random distribution of [PSI+] propagons in both vegetative (mitotic) and sporulation (meiotic) divisions  mutants and heterozygotes formed between them and with strains expressing wild type Sup35, and also in partially dominant PNM1(HSP104) mutants. Such prion instability characteristically appears as sectoring in colonies growing on normal growth medium (Fig. Another question we raise in our new study (Ness et al. ium Fig. .PSI+] prions is due to significant degradation or disaggregation of the amyloid form. Disaggregation can be observed in vitro (Shorter and Lindquist What is not certain is that any form of intrinsic or induced instability of Sup35/ Sup35 amyloid aggregates. This leaves us with the need to find other explanations for the appearance of prion-free cells.One of the epidemiological myths about the mammalian PrPr et al.  could nor et al. . Fragmenr et al. . In spit


The dismal prognosis of glioblastoma is, at least in part, attributable to the difficulty in eradicating glioblastoma stem cells (GSCs). However, whether this difficulty is caused by the differential responses of GSCs to drugs remains to be determined. To address this, we isolated and characterized ten GSC lines from established cell lines, xenografts, or patient specimens. Six lines formed spheres in a regular culture condition, whereas the remaining four lines grew as monolayer. These adherent lines formed spheres only in plates coated with poly-2-hydroxyethyl methacrylate. The self-renewal capabilities of GSCs varied, with the cell density needed for sphere formation ranging from 4 to 23.8 cells/well. Moreover, a single non-adherent GSC either remained quiescent or divided into two cells in four-seven days. The stem cell identity of GSCs was further verified by the expression of nestin or glial fibrillary acidic protein. Of the two GSC lines that were injected in immunodeficient mice, only one line formed a tumor in two months. The protein levels of NOTCH1 and platelet derived growth factor receptor alpha positively correlated with the responsiveness of GSCs to γ-secretase inhibitor IX or imatinib, two compounds that inhibit these two proteins, respectively. Furthermore, a combination of temozolomide and a connexin 43 inhibitor robustly inhibited the growth of GSCs. Collectively, our results demonstrate that patient-derived GSCs exhibit different growth rates in culture, possess differential capabilities to form a tumor, and have varied responses to targeted therapies. Our findings underscore the importance of patient-derived GSCs in glioblastoma research and therapeutic development. Glioblastoma (GBM) is the most common malignancy in the central nervous system and accounts for more than 45% of all malignant brain tumors . The proOne important obstacle against effective treatment is the intra and inter-tumoral heterogeneity within GBMs, which is predominantly caused by the presence of self-renewing GBM stem cells (GSCs) –8. GSCs While prospectively distinguishing GSCs, which reside at the apex of tumor hierarchies, from their differentiated progeny remains challenging, the similarity of GSC to its normal counterpart neural stem cell has greatly helped identification and isolation of GSCs from bulk tumors –17. Of nin vitro [Many targeted therapies have been developed to eliminate GSCs. Among these therapeutic targets, NOTCH1 –31 and Pin vitro , 29, 30.in vitro , 38–40. in vitro . TMZ is in vitro , 43. By in vitro . NonetheTo target individual GSCs, we isolated 10 GSC lines from established cell lines, xenografts, and freshly dissected tumor tissues. We then characterized these lines and verified their stem cell identity and tumorigenicity. Furthermore, we measured the effect of different cancer drugs on these cells. Our results will facilitate the development of new and effective therapies to eradicate GSCs.GSCs have been previously isolated from established GBM cell lines as well as primary tumor tissues using sphere formation or CD133-based cell sorting , 44–47. in vitro and to form tumors in vivo.To further characterize GSCs, we monitored the self-renewal of these lines. Self-renewal is determined by the capacity of GSCs to copy themselves . We usedGSCs, similar to normal stem cells, tend to divide slowly and often keep quiescent. We then monitored the growth of single cells from three non-adherent GSC lines VTC-034/GSC, VTC-064/GSC, and GBM10/GSC. We found that some single GSCs remained at one-cell stage for 7 days, suggesting that they were quiescent Figure , and 3E.2) were 0.03 or 0.06, respectively. Hence, the expression levels of marker proteins in GSC lines derived from GBM patients can only be used to verify their stem cell identity, but not the propensity to proliferate. While PolyHEMA helps establishment of spheres, this agent affects stem cell growth, differentiation, and perhaps tumor formation. Cautions should be taken to interpret data from adherent GSCs cultured in polyHEMA-coated plates.We next sought to determine the expression levels of a neural stem cell marker nestin (NES) and an astrocyte marker glial fibrillary acidic protein (GFAP) in GSCs using immunoblotting. We found that NES was expressed in GSC lines at different levels Figure . The relP values were 0.0085 or 0.019, respectively), whereas PDGFRA levels were similar . However, γ-secretase inhibitor IX had no effect on cell viability  models are superior to xenograft models from established cells lines in capitulating the pathobiology of GBM, particularly tumor heterogeneity –51. Thist tumors , 50. Thet tumors , 52, 54.PDX models could be used for drug screening, which is supported by our finding that the expression levels of PDGFRA or NOTCH1 positively correlate with the susceptibility of individual GSCs to imatinib and γ-secretase inhibitor IX. Using this initial screening could decrease the large failure rates of drugs that reach phase III clinical trails . In addiBased on the results described, we conclude that GSCs isolated from different individual tumors have discrete capabilities to grow in culture and that the expression levels of therapeutic targets in individual GSCs determine the effectiveness of corresponding targeted therapies. Our results demonstrate that the GSC lines we have established are suitable for testing the efficacy of cancer drugs, which will facilitate the discovery of GSC-eradicating drugs and provide cell models for the development of precision medicine for GBM patients.® B-27® Supplements (Life Technologies Corporation), 20 ng/ml FGF-2 (GenScript), and 20 ng/ml EGF (GenScript). LN229/GSCs, VTC-037/GSCs, VTC-061/GSCs, and VTC-084/GSCs were either grown as monolayer in above stem cell media or as spheres in flasks coated with poly(2-hydroxyethyl methacrylate) .Human GBM cell lines U251 and LN229 and primary cells isolated from GBM10 xenograft were maintained in Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum , streptomycin (100 μg/ml), and penicillin (100 IU/ml). Human GSC lines were maintained as spheres in stem cell media, which include DMEM, GibcoInstitutional Review Board at the Carilion Clinic has approved the use of human GBM patient specimens. GSCs were isolated as described previously with modifications , 44–47. ax+b) was applied to calculate the number of cells required for sphere formation (abbreviated as sphere formation number). The following formula was used: sphere formation number (x-intercept) = –b/a. The constants a and b were determined by the linear regression model.Sphere-formation assays were performed as described earlier , 57. BriIn some experiments, GSCs selected for drug treatment were subject to a modified self-renewal assay. GSCs were plated in a 96 well plate at cell densities from 10 to 80 cells per well. GSCs were treated with DMSO, 10 μM of imatinib, or 20 μM of γ-secretase inhibitor IX. After 2 weeks the number of wells with spheres was counted and the sphere pictures were taken using an inverted microscope with a 40X lens. In one of the drug treatment experiments, GSCs were plated into a 96 well plate at a cell density of 50 cells per well. GSCs were treated with vehicle, 50 μM of TMZ, 100 μM of αCT1, or a combination of TMZ and αCT1 αCT1 treatment was repeated every fourth day for 2 doses.To analyze single GSCs, one GSC was plated in a well of a 96-well plate. The cell was cultured in stem cell media and imaged using a Zeiss inverted microscope every day for 7 consecutive days.Immunoblotting was performed as described in our previous reports , 56, 58.3 GSCs were plated in a 96-well plate. Cells were then treated with DMSO, 50 μM of TMZ, 100 μM of αCT1, or a combination of TMZ and αCT1. αCT1 treatment was repeated every fourth day for 2 doses. In another set of experiments, cells were treated with imatinib or γ-secretase inhibitor IX as described above. After one week, cell viability was monitored using the MTS assay. 10 μl of MTS (Promega) was added to each well, then incubated at 37°C for 1 h. The absorbance at 490 nm was measured using a FilterMax F3 microplate reader  according to manufacturer's instructions. Percent cell viability was obtained by dividing the absorbance of treatment groups with those of untreated groups.MTS viability assays were described previously , 58. In 5 of VTC-036/GSCs or VTC-037/GSCs were subcutaneously injected into SCID/beige mice . A tumor from VTC-036/VTCs with a diameter of 1 cm formed in 68 days, whereas no tumor was found in the mouse receiving VTC-037/GSCs. The tumor was harvested and stained by Hematoxylin and Eosin.Mouse experiments were performed based on methods described previously, with modifications , 58. Allt test and one-way ANNOVA were used to determine the difference among treatment groups.Student's


Biochemically, ARQ 087 exhibited IC50 values of 1.8 nM for FGFR2, and 4.5 nM for FGFR1 and 3. In cells, inhibition of FGFR2 auto-phosphorylation and other proteins downstream in the FGFR pathway  was evident by the response to ARQ 087 treatment. Cell proliferation studies demonstrated ARQ 087 has anti-proliferative activity in cell lines driven by FGFR dysregulation, including amplifications, fusions, and mutations. Cell cycle studies in cell lines with high levels of FGFR2 protein showed a positive relationship between ARQ 087 induced G1 cell cycle arrest and subsequent induction of apoptosis. In addition, ARQ 087 was effective at inhibiting tumor growth in vivo in FGFR2 altered, SNU-16 and NCI-H716, xenograft tumor models with gene amplifications and fusions. ARQ 087 is currently being studied in a phase 1/2 clinical trial that includes a sub cohort for intrahepatic cholangiocarcinoma patients with confirmed FGFR2 gene fusions (NCT01752920).Dysregulation of Fibroblast Growth Factor Receptor (FGFR) signaling through amplifications, mutations, and gene fusions has been implicated in a broad array of cancers . ARQ 087 is a novel, ATP competitive, small molecule, multi-kinase inhibitor with potent  The FGFR family of tyrosine kinase receptors FGFR1-4), and their ligands, the fibroblast growth factors (FGFs), play an important role in multiple signal transduction pathways including mitogen-activated protein kinases (MAPK), phosphatidylinositol 3-kinase (PI3K) phospholipase Cγ (PLCγ), protein kinase C (PKC), and signal transducers and activator of transcription (STAT). FGFR activation leads to a series of cellular signaling events including increased cellular proliferation, differentiation, and migration -4, and t.in vitro and in vivo has been shown to reduce proliferation of FGFR-dependent cancer cells and induces cell death  x 100.Six week old female NCr nu/nu mice (SNU-16) or CB17 SCID mice (NCI-H716) with well established (~400 mg) subcutaneous tumors were given a single oral dose of ARQ 087 or vehicle control. Plasma and tumor samples were collected 4 hours post single dose. For all in vivo experiments, ARQ 087 was formulated in DMA: cremophor EL: propylene glycol: 0.2 M acetate buffer, pH 5 (10:10:30:50) and administered orally. Dosing volume for all groups was 10 mL/kg or 0.1 mL/10 g body weight.For in vivo pathway inhibition studies, female NCr Xenograft tumor tissues were collected after the 4-hour drug treatment, fixed for 16–24 hours in 10% neutral buffered formalin (NBF) and embedded in paraffin. Immunohistochemistry (IHC) was performed on the tumor tissue sections (5 μm thickness) for a panel of biomarkers. After deparaffination and rehydration, antigen retrieval was conducted in a decloaking chamber for 30 minutes at 95°C, followed by 10 seconds at 90°C using a 1 μM EDTA solution pH 8.0  for total FGFR2, phospho-FGFR, and phospho-FRS2α; 10 mM Citrate buffer pH 6.0  for phospho-ERK. IHC was performed using an autostainer 480 (Lab vision UK Ltd). These antibodies were diluted in 1% BSA/TBST. The sections were incubated with primary antibody for 30 minutes and then with secondary antibody (or the polymer) for 25 minutes at ambient temperature. Rabbit on Rodent HRP-Polymer  or Goat anti-rabbit HRP  was used as a secondary antibody. 3,3’-diaminobenzidine (DAB) was used as the chromogen and the slides were counter-stained with hematoxylin after IHC.in vivo studies, all statistical analyses were performed using an unpaired t test. Data were presented as mean ± SEM. A p value of less than 0.05 was considered to be significant.For in silico design and characterization of 5, 6 dihydrobenzo[h]quinazolin-2-amine as a novel chemical series of FGFR kinase inhibitors quinazolin-2-yl)amino)phenethyl methanesulfonate which was then subjected to the displacement of the the methanesulfonate by 2-methoxyethylamine in presence of trimethylamine, giving after acidic work up, (R)-6-(2-fluorophenyl)-N-(3-(2-((2-methoxyethyl)amino)ethyl)phenyl) -5,6-dihydrobenzo[h]quinazolin-2-amine (ARQ 087) as a HCl salt.ARQ 087 is prepared as follows : from  and the Bayesian information criterion (BIC) to determine the best fitting model. The ATP competitive model produced the lowest AIC and BIC values, indicating that ARQ 087 is a competitive inhibitor of both FGFR1 and FGFR2 Ki values of FGFR1 and FGFR2 were determined to be 2.7 ± 0.2 nM and 0.68 ± 0.07 nM, respectively, demonstrating a strong inhibitory potency of ARQ 087 for FGFR1 and FGFR2 ARQ 087 inhibited wild-type FGFR1, FGFR2, and FGFR3 with biochemical ICActivation of FGFR kinases requires auto-phosphorylation on multiple tyrosine residues , 40. Inh50 values of this subset of kinases revealed that 18 wild-type kinases (excluding FGFRs), exhibited sensitivities to ARQ 087 within a 3- to 10-fold range of the IC50 value for FGFR2 The selectivity of ARQ 087 across the kinome, was evaluated against a panel of 298 kinases at a concentration of 0.1 μM. Among 298 kinases assayed (kinase domain), approximately 50  were inhibited by greater than 50% by ARQ 087. Examination of the IC50 values of < 0.123 μM, 0.185 μM, 0.463 μM, >10 μM respectively Inhibition of FGFRs in transfected cell lines: To determine whether ARQ 087 inhibits the phosphorylation of FGFR1, FGFR2, FGFR3, and FGFR4 in cells, we over-expressed full-length FGFR1, FGFR2, FGFR3 and FGFR4 in COS-1 cells and examined the effect of ARQ 087 on phosphorylation of FGFRs . We found that ARQ 087 inhibited the phosphorylation of FGFR1, FGFR2, FGFR3, and FGFR4 with EC50 values for Ba/F3-FGFR1, Ba/F3-FGFR2, Ba/F3-FGFR3, and Ba/F3-FGFR4 ranged between 232 nM and 1346 nM, with Ba/F3-FGFR2 the most sensitive cell line to ARQ 087 followed by Ba/F3-FGFR1 and Ba/F3-FGFR3 50 values were between 39.9 nM and 1121 nM, with FGFR3-BAIAP2L1 as the most sensitive cell line We also examined the anti-proliferative effect of ARQ 087 in a panel of Ba/F3 cell lines that had been engineered to be dependent on individually over expressed tyrosine protein kinases for survival, including cell lines dependent on individual FGFR isoforms and FGFR fusions. The GI50 values above 1000 nM, with the exception of ARG, LCK, KIT, FGFR1-2, and FGFR fusions as shown in In addition to FGFR-driven models, 15 other kinases that were potently inhibited by ARQ 087 biochemically were also investigated in the Ba/F3 engineered cell lines. These data reinforce the biochemical data that suggests ARQ 087 does inhibit kinases outside of the FGFR family in cellular assays. However, the majority of kinases had GIFGFR pathway inhibition was examined in three solid tumor lines, the colorectal cancer cell line (NCI-H716), and gastric cancer cell lines (SNU-16 and KATO-III), all these cell lines are FGFR2 amplified , 42, 43.We next examined the signal transduction pathways activated downstream of FGFR in three FGFR2-dependent  cancer cell lines. ARQ 087 inhibited the phosphorylation of FGFR2 and its immediate downstream substrate, FRS2α, as well as the phosphorylation of further downstream components MEK, ERK and AKT In addition, we also tested ARQ 087 in the acute myeloid leukemia cell line KG-1. This cell line is driven FGFR1 fusion gene, FGFR1OP-FGFR1 . Here weWe then evaluated the anti-proliferative effect of ARQ 087 in a number of cell lines that included cells with amplified FGFR, mutant FGFR, translocated FGFR, or unknown FGFR status. ARQ 087 demonstrated good potency in cell lines with FGFR2 amplifications and fusions. NCI-H716, SNU-16, and KG-1, were some of the most sensitive cell lines in this panel 50 values between 0.13 to 1.4 μM ARQ 087 inhibited multiple cell lines known to have FGFR1 or FGFR3 fusions including; RT4 and RT112 (FGFR3-TACC3), SW-780 (FGFR3-BAIAP2L1), and KG-1(FGFROP2-FGFR1), with GIThe mechanism by which ARQ 087 inhibits the growth of FGFR kinase dependent cell lines was investigated. NCI-H716 and SNU-16 cells were treated with either 0.1 μM or 1 μM of ARQ 087 for 24 or 72 hours then subjected to flow cytometric analysis. Camptothecin, a known inhibitor of cell cycle progression was used as the positive control in these experiments. Treatment with ARQ 087 led to an accumulation of cells in the G1 phase cell cycle in a dose- and time-dependent manner in vivo pharmacodynamic effect of ARQ 087 was examined in SNU-16 tumor-bearing animals using IHC. Doses of 0, 25, 50, and 75 mg/kg of ARQ 087 (n = 9/group) led to a reduction in phospho-FGFR, phospho-FRS2-α, and phospho-ERK after 4 hours, while the total FGFR2 protein was unaffected by ARQ 087 treatment The in vivo anti-tumor effect of ARQ 087 was assessed in athymic mice bearing Ba/F3-FGFR2, Ba/F3-INSR, SNU-16, and NCI-H716 cell line-derived tumors. The Ba/F3-FGFR2 and Ba/F3-INSR are transfected models. The SNU-16 cell line harbors amplified FGFR2 and contains a PDHX-FGFR2 fusion, while the NCI-H716, also amplified for FGFR2, contains a FGFR2-COL14A1 fusion. ARQ 087 demonstrated potent tumor growth inhibition in the Ba/F3-FGFR2 model, while failing to inhibit the growth of the Ba/F3-INSR model The in vitro cell models with a genetically dysregulated FGFR2 pathway.Data presented here clearly demonstrate the preclinical and clinical activity of ARQ 087, a potent pan-FGFR inhibitor showing multi-kinase activity. ARQ 087 inhibits FGFR kinase by an ATP competitive mechanism, and is capable of inhibiting both the inactive and fully active forms of the FGFR kinase. Hence, ARQ 087 delays FGFR activation by inhibiting its autophosphorylation, as well as inhibition of the phosphorylated active kinase. These data prompted further investigation of ARQ 087 using In cellular models, ARQ 087 was shown to inhibit FGFR phosphorylation in ectopically-expressing COS-1 cells with essentially equivalent potency for FGFR1 and FGFR2. Additionally, ARQ 087 inhibited FGFR phosphorylation in FGFR2 dependent cells. FGFR2 pathway inhibition by ARQ 087 was confirmed in multiple FGFR2 dependent cell lines further supporting FGFR2 as a target of ARQ 087.The functional consequence of FGFR inhibition was assessed in multiple cell systems. ARQ 087 inhibited cell proliferation in Ba/F3 transfected cells which were engineered to be dependent on FGFR1-4 signaling, or on an FGFR gene fusion.Among the cell lines that harbored FGFR fusions, amplifications, or mutations, cell lines with FGFR-fusions were among the most sensitive to ARQ 087. Likewise, Ba/F3 cell lines addicted to FGFR-fusions profiled better than the wild-type or mutant FGFR dependent Ba/F3 cells. Interestingly, a similar observation has been reported by Arai et al  and Wu eFurther investigation of the mechanism of growth inhibition in FGFR-driven tumor cell lines demonstrated that ARQ 087 induced an accumulation of cells in G1 phase of the cell cycle. Further studies suggested that ARQ 087 works through an apoptotic mechanism of cell death in the SNU-16 cell line which is FGFR2 amplified as well as harboring FGFR2 fusions.In vivo assessment of pharmacodynamic biomarkers four hours after a single dose of ARQ 087 in animals with FGFR2 driven tumors showed a reduction in the phosphorylation of both FGFR and its substrate FRS2-α. Additionally the downstream marker phospho-ERK, was also shown to decrease following ARQ 087 treatment. In FGFR2-amplified tumor models where highly activated MAPK signaling (phospho-ERK staining) is driven by FGFR2 signaling, the downstream markers are also strongly inhibited by ARQ 087. These data agree well with the in vitro data demonstrating inhibition of the FGFR signaling pathway in these cell lines and induction of an apoptotic response.in vivo xenograft models, and was well tolerated at doses up to 75 mg/kg. Durable partial and complete regressions were observed in both tumor models that are driven by FGFR2 amplification and fusions (NCI-H716 and SNU-16), and the murine transfected cell line (BaF3/FGFR2). Notably, ARQ 087 was not efficacious in a xenograft model without dependency on FGFR2: i.e. BaF3/INSR (BaF3 cells expressing the insulin receptor).ARQ 087 demonstrated efficacy in multiple There are a number of FGF/FGFR targeted therapies currently in preclinical or clinical development, including small molecule non-selective FGFR multi-kinase inhibitors, selective FGFR inhibitors, FGFR antibodies, and FGF ligand traps –17. AlthThe recent identification of the driver role of FGFR2 fusions in iCCA, as well as other tumor types, provides a promising path forward in the selection of patients that may respond to FGFR inhibitors in a clinical setting , 56. Thein vitro and in vivo. ARQ 087 has recently completed the dose escalation part of its first in human clinical trial [NCT01752920]. A clinical response was noted for a patient with intrahepatic cholangiocarcinoma with a known FGFR2 fusion, additionally prolonged stable disease was observed in 10 of 61 patients, including a cholangiocarcinoma, which also had an FGFR2 fusion [In conclusion, ARQ 087 is a novel and potent FGFR inhibitor with multi-kinase activity. It has the potential to be effective in variety of cancers driven by dysregulation of the FGFR pathway. Preclinical results suggest that genetic alterations of the FGFR pathway—amplification, mutation, and gene fusion, correlate with the activity of ARQ 087 2 fusion . Full reS1 Appendix(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.