Search

Supplementary Materials and Methods, Figures S1-5 and Tables S1-8. Supplementary Materials and Methods: More detailed description of Materials and Methods for experiments. Figure S1: Structural analysis of the FGFR3-IIIb and R3Mab complex. Figure S2: Sequence analysis of the CDR H2 of selected FGFR2-positive clones. Figure S3: Superimposition of crystal structures of FGFR2-D2:2B.1.3 and FGFR3-D2D3:R3Mab. Figure S4: in vitro and in vivo studies of the 2B.1 antibody variants using the breast cancer cell line MFM-223x2.2. Figure S5: FGFR2 and FGFR3 expressions in gastric cancer cell line SNU-16. Table S1: Library design for recruiting FGFR2 binding specificity for R3Mab. Table S2: Library design for removing FGFR4 binding specificity from 2B.1.3. Table S3: Sequence identities between human FGFR. Table S4: Bidning affinities of selected 2B.1 variants for FGFR2 and FGFR3. Table S5: X-ray crystallography data collection and refinement statistics. Table S6: Binding affinities of R3Mab and 2B.1.3 for the D2 alone and D2D3 constructs of FGFR2 and FGFR3. Table S7: Residue differences between FGFR2 and FGFR4 at the positions making potential contacts with 2B.1.3. Table S8: 2B.1.3 variants that retain FGFR2 binding and show diminished binding to FGFR4.

R3Mab reduces MMP protein secretion in the conditioned medium by UMUC-14 cells.

Supplementary Fig. S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Cancer cells exploit the unfolded protein response (UPR) to mitigate endoplasmic reticulum (ER) stress caused by cellular oncogene activation and a hostile tumor microenvironment (TME). The key UPR sensor IRE1α resides in the ER and deploys a cytoplasmic kinase–endoribonuclease module to activate the transcription factor XBP1s, which facilitates ER-mediated protein folding. Studies of triple-negative breast cancer (TNBC)—a highly aggressive malignancy with a dismal posttreatment prognosis—implicate XBP1s in promoting tumor vascularization and progression. However, it remains unknown whether IRE1α adapts the ER in TNBC cells and modulates their TME, and whether IRE1α inhibition can enhance antiangiogenic therapy—previously found to be ineffective in patients with TNBC. To gauge IRE1α function, we defined an XBP1s-dependent gene signature, which revealed significant IRE1α pathway activation in multiple solid cancers, including TNBC. IRE1α knockout in TNBC cells markedly reversed substantial ultrastructural expansion of their ER upon growth in vivo. IRE1α disruption also led to significant remodeling of the cellular TME, increasing pericyte numbers while decreasing cancer-associated fibroblasts and myeloid-derived suppressor cells. Pharmacologic IRE1α kinase inhibition strongly attenuated growth of cell line–based and patient-derived TNBC xenografts in mice and synergized with anti-VEGFA treatment to cause tumor stasis or regression. Thus, TNBC cells critically rely on IRE1α to adapt their ER to in vivo stress and to adjust the TME to facilitate malignant growth. TNBC reliance on IRE1α is an important vulnerability that can be uniquely exploited in combination with antiangiogenic therapy as a promising new biologic approach to combat this lethal disease.Significance:Pharmacologic IRE1α kinase inhibition reverses ultrastructural distension of the ER, normalizes the tumor vasculature, and remodels the cellular TME, attenuating TNBC growth in mice.

Supplementary Table S1 from Antixenograft tumor activity of a humanized anti-insulin-like growth factor-I receptor monoclonal antibody is associated with decreased AKT activation and glucose uptake

Supplementary Data, including Supplementary Materials and Methods, References for Supplementary Data, and Figs. S1 to S5 including Figure Legends

List of patient information in the phase I study.

The insulin-like growth factor (IGF) system consists of two ligands (IGF-I and IGF-II), which both signal through IGF-I receptor (IGF-IR) to stimulate proliferation and inhibit apoptosis, with activity contributing to malignant growth of many types of human cancers. We have developed a humanized, affinity-matured anti-human IGF-IR monoclonal antibody (h10H5), which binds with high affinity and specificity to the extracellular domain. h10H5 inhibits IGF-IR-mediated signaling by blocking IGF-I and IGF-II binding and by inducing cell surface receptor down-regulation via internalization and degradation, with the extracellular and intracellular domains of IGF-IR being differentially affected by the proteasomal and lysosomal inhibitors. In vitro, h10H5 exhibits antiproliferative effects on cancer cell lines. In vivo, h10H5 shows single-agent antitumor efficacy in human SK-N-AS neuroblastoma and SW527 breast cancer xenograft models and even greater efficacy in combination with the chemotherapeutic agent docetaxel or an anti–vascular endothelial growth factor antibody. Antitumor activity of h10H5 is associated with decreased AKT activation and glucose uptake and a 316-gene transcription profile with significant changes involving DNA metabolic and cell cycle machineries. These data support the clinical testing of h10H5 as a biotherapeutic for IGF-IR-dependent human tumors and furthermore illustrate a new method of monitoring its activity noninvasively in vivo via 2-fluoro-2-deoxy-d-glucose-positron emission tomography imaging. [Mol Cancer Ther 2008;7(9):2599–608]

R3Mab attenuates xenograft growth of UMUC-14 tumors.

Purpose: The aim of this study was to identify noninvasive pharmacodynamic biomarkers of FGFR3-targeted therapies in bladder cancer to facilitate the clinical development of experimental agent targeting FGFR3.Experimental Design: Potential soluble pharmacodynamic biomarkers of FGFR3 were identified using a combination of transcriptional profiling and biochemical analyses in preclinical models. Two matrix metalloproteinases (MMP), MMP-1 and MMP-10, were selected for further studies in human bladder cancer xenograft models treated with a specific anti-FGFR3 monoclonal antibody, R3Mab. Serum and urinary levels of MMP-1 and MMP-10 were determined in healthy donors and patients with bladder cancer. The modulation of MMP-1 and MMP-10 by R3Mab in patients with bladder cancer was further evaluated in a phase I dose-escalation study.Results: MMP-1 and MMP-10 mRNA and protein were downmodulated by FGFR3 shRNA and R3Mab in bladder cancer cell lines. FGFR3 signaling promoted the expression and secretion of MMP-1 and pro-MMP-10 in a MEK-dependent fashion. In bladder cancer xenograft models, R3Mab substantially blocked tumor progression and reduced the protein levels of human MMP-1 and pro-MMP-10 in tumor tissues as well as in mouse serum. Furthermore, both MMP-1 and pro-MMP-10 were elevated in the urine of patients with advanced bladder cancer. In a phase I dose-escalation trial, R3Mab administration resulted in an acute reduction of urinary MMP-1 and pro-MMP-10 levels in patients with bladder cancer.Conclusion: These findings reveal a critical role of FGFR3 in regulating MMP-1 and pro-MMP-10 expression and secretion, and identify urinary MMP-1 and pro-MMP-10 as potential pharmacodynamic biomarkers for R3Mab in patients with bladder cancer. Clin Cancer Res; 20(24); 6324–35. ©2014 AACR.

Contains a list of genes regulated by FGFR3 shRNA in RT112 cells.

R3Mab reduces serum levels of human MMP-1 and pro-MMP-10 in mice bearing bladder tumor xenografts.

FGFR3 knockdown reduces MMP-1 and MMP-10 mRNA expression in RT112 cells.

Supplementary Data from Development of Immunohistochemistry Assays to Assess GALNT14 and FUT3/6 in Clinical Trials of Dulanermin and Drozitumab

FGFR3 knockdown in RT112 cells reduces total MMP-10 protein level in conditioned medium.

Purpose: In vitro sensitivity to the proapoptotic receptor agonists dulanermin (rhApo2L/TRAIL) and drozitumab (DR5-agonist antibody) is strongly predicted by the expression of the O-glycosylation enzymes GALNT14 in non–small cell lung cancer (NSCLC) cell lines (among others) and of FUT3/6 in colorectal cancer (CRC) cell lines. We developed immunohistochemistry (IHC) assays that measure GALNT14 and FUT3/6 levels in archival formalin-fixed, paraffin-embedded human tumor tissue to determine marker prevalence in NSCLC and CRC tissue and to enable the future examination of these markers in clinical trials.Experimental Design: GALNT14 or FUT3/6 ELISA-positive hybridoma clones were screened through IHC on cell pellets with known mRNA levels. The specificity of staining was examined in cell lines, normal tissue, and tumor tissue.Results: GALNT14 and FUT3/6 IHC exhibited a golgi staining pattern and correlated with GALNT14 and FUT3/6 (but not GALNT2 and FUT4) mRNA expression levels in cell lines and normal tissues, suggesting specificity. GALNT14 and FUT3/6 H-scores were significantly higher in cell lines sensitive to dulanermin (P = 0.01 and P = 0.0004, respectively) and drozitumab (P = 0.03 and P < 0.0001, respectively) versus resistant cell lines. GALNT14 and FUT3/6 H-scores varied widely, with ∼45% of NSCLC samples exhibiting weak to moderate GALNT14 staining (H-score of at least 25) and 70% of CRC samples exhibiting moderate to strong FUT3/6 staining (H-score of at least 125).Conclusions: GALNT14 and FUT3/6 expression can be assessed in human tumors using sensitive and specific IHC assays. Both assays are being deployed in ongoing clinical trials of dulanermin and drozitumab to assess potential utility for patient selection. Clin Cancer Res; 16(5); 1587–96

Supplemental Dataset 1. Expression of AXL, GAS6, Vimentin and E-Cadherin in 644 human cancer cell line using RNAseq technology.

Timelapse movie of HeLa cells treated with R428 (1μM).

Timelapse movie of HeLa cells treated with DMSO.

PDF file, 114KB, This section includes Supplemental Materials and Methods. It also contains the legends to Supplemental Figures 1-9.

Timelapse movie of HeLa cells treated with docetaxel (3nM).

Supplemental Dataset 3. Tumor cell line profile of docetaxel in combination with R428

Supplemental Dataset 2. Kinome profile with 1000nM R428

Timelapse movie of HeLa cells treated with docetaxel (3nM) in combination with R428 (1μM).

XLS file - 97KB, List of genes that showed significant changes upon FGFR3 knockdown.

PDF file - 800KB, Figure S1 Identification of FGFR3-regulated genes. Figure S2 FGFR3 knockdown in RT112 bladder cancer cells reduces the expression of genes involved in sterol and fatty acid biosynthesis and metabolism. Figure S3 Inhibition of FGFR3 reduces the expression of genes involved in sterol and fatty acid biosynthesis and metabolism in UMUC-14 cells. Figure S4 FGF1 stimulates SREBP1 activation and lipogenesis in bladder cancer cells. Figure S5 FGFR3 signaling promotes the accumulation of matured SREBP1 and SCD1 expression via PI3K-mTORC1 pathway but not MEK-MAPK pathway in Cal29 cells. Figure S6 SCD1 knockdown induces apoptosis in bladder cancer cells. (A) FGFR3 signaling in SW780 bladder cancer cells. Figure S7 SCD1 knockdown inhibits bladder cancer cell proliferation in a fatty acid desaturation-dependent manner. Figure S8 SCD1 inhibitor suppresses cell proliferation in a fatty acid desaturation-dependent manner in SW780 cells. Figure S9 SCD1 inhibitor A37062 activates caspase 3 in UMUC-14 xenograft tumors.Supplemental Table 2 Mutational status of FGFR3 and pathway activity in bladder cancer cell lines

Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target., The RNase activity of Inositol-Requiring Enzyme 1 (IRE1) can be allosterically regulated by ATP-competitive inhibitors of the IRE1 kinase domain. Here, the authors identify ATP-competitive IRE1 RNase activators with improved selectivity and cellular activity, and elucidate their mechanism of action.

Dendritic cells (DCs) promote adaptive immunity by cross-presenting antigen-based epitopes to CD8+ T cells. DCs process internalized protein antigens into peptides that enter the endoplasmic reticulum (ER), bind to major histocompatibility type I (MHC-I) protein complexes, and are transported to the cell surface for cross-presentation. DCs can exhibit activation of the ER stress sensor IRE1α without ER stress, but the underlying mechanism remains obscure. Here, we show that antigen-derived hydrophobic peptides can directly engage ER-resident IRE1α, masquerading as unfolded proteins. IRE1α activation depletes MHC-I heavy-chain mRNAs through regulated IRE1α-dependent decay (RIDD), curtailing antigen cross-presentation. In tumor-bearing mice, IRE1α disruption increased MHC-I expression on tumor-infiltrating DCs and enhanced recruitment and activation of CD8+ T cells. Moreover, IRE1α inhibition synergized with anti–PD-L1 antibody treatment to cause tumor regression. Our findings identify an unexpected cell-biological mechanism of antigen-driven IRE1α activation in DCs, revealing translational potential for cancer immunotherapy.

We report results from a search for neutrino-induced neutral current (NC) resonant $\Delta$(1232) baryon production followed by $\Delta$ radiative decay, with a $\langle0.8\rangle$~GeV neutrino beam. Data corresponding to MicroBooNE's first three years of operations (6.80$\times$10$^{20}$ protons on target) are used to select single-photon events with one or zero protons and without charged leptons in the final state ($1\gamma1p$ and $1\gamma0p$, respectively). The background is constrained via an in-situ high-purity measurement of NC $\pi^0$ events, made possible via dedicated $2\gamma1p$ and $2\gamma0p$ selections. A total of 16 and 153 events are observed for the $1\gamma1p$ and $1\gamma0p$ selections, respectively, compared to a constrained background prediction of $20.5 \pm 3.65 \text{(sys.)} $ and $145.1 \pm 13.8 \text{(sys.)} $ events. The data lead to a bound on an anomalous enhancement of the normalization of NC $\Delta$ radiative decay of less than $2.3$ times the predicted nominal rate for this process at the 90% confidence level (CL). The measurement disfavors a candidate photon interpretation of the MiniBooNE low-energy excess as a factor of $3.18$ times the nominal NC $\Delta$ radiative decay rate at the 94.8% CL, in favor of the nominal prediction, and represents a greater than $50$-fold improvement over the world's best limit on single-photon production in NC interactions in the sub-GeV neutrino energy range

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1's lumenal domain. Phosphorylation of IRE1's kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enablesOur cells contain many different compartments that each perform specific tasks. A cellular compartment known as the endoplasmic reticulum is responsible for making many of the proteins the cell requires and transporting them around the cell. It is important that the endoplasmic reticulum remains healthy and, therefore, cells use a protein called IRE1 that senses when this compartment is under stress. IRE1 then sends a signal to the control center of the cell (known as the nucleus) to ask for help. Previous studies suggest that IRE1 assembles into either pairs or larger groups of molecules known as oligomers when it senses that the endoplasmic reticulum is under stress. However, it remains unclear whether such assembly is the main switch that turns IRE1 on and, if so, how many molecules need to come together to flip the switch. Here, Belyy et al. genetically engineered human bone cancer cells to attach a mark known as a HaloTag to IRE1.The team developed a microscopy approach to count, in living cells, how many tagged IRE1 molecules join. The experiments indicated that IRE1 proteins were generally found as pairs in unstressed cells. When the endoplasmic reticulum experienced stress, IRE1 proteins briefly assembled into oligomers before disassembling back into pairs. Mutated versions of IRE1 revealed the exact parts of IRE1 that connect the pairs and the larger oligomers. These findings suggest that the assembly of IRE1 pairs into oligomers plays a major part in the activation of IRE1 to send a stress signal to the nucleus. IRE1 signaling is closely implicated in both cancer biology and aging, and therefore, understanding how it works may aid the development of new therapies for cancer, dementia, and other health conditions affecting older people. Furthermore, the microscopy approach developed in this work could be adapted to study other proteins that relay signals in living cells.

This document was prepared by the MicroBooNE collaboration using the resources of the Fermi National Accelerator Laboratory (Fermilab), a U.S. Department of Energy, Office of Science, HEP User Facility. Fermilab is managed by Fermi Research Alliance, LLC (FRA), acting under Contract No. DE-AC02-07CH11359. MicroBooNE is supported by the following: the U.S. Department of Energy, Office of Science, Offices of High Energy Physics and Nuclear Physics; the U.S. National Science Foundation; the Swiss National Science Foundation; the Science and Technology Facilities Council (STFC), part of the United Kingdom Research and Innovation; and The Royal Society (United Kingdom). Additional support for the laser calibration system and cosmic ray tagger was provided by the Albert Einstein Center for Fundamental Physics, Bern, Switzerland. Participation of individual researchers in this project is supported by funds from the European Unions Horizon 2020 Research and Innovation Programme under the Marie SklodowskaCurie Grant Agreement No. 752309. We thank Brian Batell, Joshua Berger, and Ahmed Ismail for outlining the search strategy., We present a search for the decays of a neutral scalar boson produced by kaons decaying at rest, in the context of the Higgs portal model, using the MicroBooNE detector. We analyze data triggered in time with the Fermilab NuMI neutrino beam spill, with an exposure of 1.93 x 10(20) protons on target. We look for monoenergetic scalars that come from the direction of the NuMI hadron absorber, at a distance of 100 m from the detector, and decay to electron-positron pairs. We observe one candidate event, with a standard model background prediction of 1.9 +/- 0.8. We set an upper limit on the scalar-Higgs mixing angle of theta < (3.3 - 4.6) x 10(-4) at the 95% confidence level for scalar boson masses in the range (100-200) MeV/c(2). We exclude, at the 95% confidence level, the remaining model parameters required to explain the central value of a possible excess of K-L(0) -> pi(0)nu(nu) over bar decays reported by the KOTO collaboration. We also provide a model-independent limit on a new boson X produced in K -> pi X decays and decaying to e(+)e(-)., Fermi Research Alliance, LLC (FRA) DE-AC02-07CH11359, United States Department of Energy (DOE), National Science Foundation (NSF), Swiss National Science Foundation (SNSF), European Commission, Science and Technology Facilities Council (STFC), part of the United Kingdom Research and Innovation, Royal Society of London, Albert Einstein Center for Fundamental Physics, Bern, Switzerland, SKA South Africa 752309

The signaling network of the unfolded protein response (UPR) adjusts the protein-folding capacity of the endoplasmic reticulum (ER) according to need. The most conserved UPR sensor, IRE1α, spans the ER membrane and activates through oligomerization. IRE1α oligomers accumulate in dynamic foci. We determined the in situ structure of IRE1α foci by cryogenic correlated light and electron microscopy combined with electron cryo-tomography and complementary immuno–electron microscopy in mammalian cell lines. IRE1α foci localized to a network of narrow anastomosing ER tubes (diameter, ~28 nm) with complex branching. The lumen of the tubes contained protein filaments, which were likely composed of arrays of IRE1α lumenal domain dimers that were arranged in two intertwined, left-handed helices. This specialized ER subdomain may play a role in modulating IRE1α signaling.

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remained unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1’s lumenal domain. Phosphorylation of IRE1’s kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans- autophosphorylation, which in turn drives IRE1’s RNase activity.

Understanding the molecular events that regulate cell pluripotency versus acquisition of differentiated somatic cell fate is fundamentally important. Studies in Caenorhabditis elegans demonstrate that knockout of the germline-specific translation repressor gld-1 causes germ cells within tumorous gonads to form germline-derived teratoma. Previously we demonstrated that endoplasmic reticulum (ER) stress enhances this phenotype to suppress germline tumor progression(Levi-Ferber et al., 2015). Here, we identify a neuronal circuit that non-autonomously suppresses germline differentiation and show that it communicates with the gonad via the neurotransmitter serotonin to limit somatic differentiation of the tumorous germline. ER stress controls this circuit through regulated inositol requiring enzyme-1 (IRE-1)-dependent mRNA decay of transcripts encoding the neuropeptide FLP-6. Depletion of FLP-6 disrupts the circuit’s integrity and hence its ability to prevent somatic-fate acquisition by germline tumor cells. Our findings reveal mechanistically how ER stress enhances ectopic germline differentiation and demonstrate that regulated Ire1-dependent decay can affect animal physiology by controlling a specific neuronal circuit.