DrFLINC Contextualizes Super-resolution Activity Imaging.

Super-resolution activity imaging maps the biochemical architecture of living cells yet currently overlooks the locations of collaborating regulators/effectors. Building on the fluorescence fluctuation increase by contact (FLINC) principle, here we devise Dronpa-chromophore-removed FLINC (DrFLINC), where the nonfluorescent Dronpa can nevertheless enhance TagRFP-T fluorescence fluctuations. Exploiting DrFLINC, we develop a superior red label and a next-generation activity sensor for context-rich super-resolution biosensing.

Metamaterial-Assisted Photobleaching Microscopy with Nanometer Scale Axial Resolution.

The past two decades have witnessed a dramatic progress in the development of novel super-resolution fluorescence microscopy technologies. Here, we report a new fluorescence imaging method, called metamaterial-assisted photobleaching microscopy (MAPM), which possesses a nanometer-scale axial resolution and is suitable for broadband operation across the entire visible spectrum. The photobleaching kinetics of fluorophores can be greatly modified via a separation-dependent energy transfer process to a nearby metamaterial. The corresponding photobleaching rate is thus linked to the distance between the fluorophores and the metamaterial layer, leading to a reconstructed image with exceptionally high axial resolution. We apply the MAPM technology to image the HeLa cell membranes tagged with fluorescent proteins and demonstrate an axial resolution of ∼2.4 nm with multiple colors. MAPM utilizes a metamaterial-coated substrate to achieve super-resolution without altering anything else in a conventional microscope, representing a simple solution for fluorescence imaging at nanometer axial resolution.

Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A protein kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membranes of living cells and to uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture whose structural and functional characteristics can be revealed by these new biosensors.

Effect of Doxorubicin Plus Olaratumab vs Doxorubicin Plus Placebo on Survival in Patients With Advanced Soft Tissue Sarcomas: The ANNOUNCE Randomized Clinical Trial.

Importance: Patients with advanced soft tissue sarcoma (STS) have a median overall survival of less than 2 years. In a phase 2 study, an overall survival benefit in this population was observed with the addition of olaratumab to doxorubicin over doxorubicin alone. Objective: To determine the efficacy of doxorubicin plus olaratumab in patients with advanced/metastatic STS. Design, Setting, and Participants: ANNOUNCE was a confirmatory, phase 3, double-blind, randomized trial conducted at 110 sites in 25 countries from September 2015 to December 2018; the final date of follow-up was December 5, 2018. Eligible patients were anthracycline-naive adults with unresectable locally advanced or metastatic STS, an Eastern Cooperative Oncology Group performance status of 0 to 1, and cardiac ejection fraction of 50% or greater. Interventions: Patients were randomized 1:1 to receive doxorubicin, 75 mg/m2 (day 1), combined with olaratumab (n = 258), 20 mg/kg in cycle 1 and 15 mg/kg in subsequent cycles, or placebo (n = 251) on days 1 and 8 for up to 8 21-day cycles, followed by olaratumab/placebo monotherapy. Main Outcomes and Measures: Dual primary end points were overall survival with doxorubicin plus olaratumab vs doxorubicin plus placebo in total STS and leiomyosarcoma (LMS) populations. Results: Among the 509 patients randomized (mean age, 56.9 years; 58.2% women; 46.0% with LMS), all were included in the primary analysis and had a median length of follow-up of 31 months. No statistically significant difference in overall survival was observed between the doxorubicin plus olaratumab group vs the doxorubicin plus placebo group in either population (total STS: hazard ratio, 1.05 [95% CI, 0.84-1.30], P = .69, median overall survival, 20.4 months vs 19.7 months; LMS: hazard ratio, 0.95 [95% CI, 0.69-1.31], P = .76, median overall survival, 21.6 months vs 21.9 months). Adverse events of grade 3 or greater reported in 15% or more of total patients with STS were neutropenia (46.3% vs 49.0%), leukopenia (23.3% vs 23.7%), and febrile neutropenia (17.5% vs 16.5%). Conclusions and Relevance: In this phase 3 clinical trial of patients with advanced STS, treatment with doxorubicin plus olaratumab vs doxorubicin plus placebo resulted in no significant difference in overall survival. The findings did not confirm the overall survival benefit observed in the phase 2 trial. Trial Registration: ClinicalTrials.gov Identifier: NCT02451943.

Systematic Modeling and Design Evaluation of Unperturbed Tumor Dynamics in Xenografts.

Xenograft mice are largely used to evaluate the efficacy of oncological drugs during preclinical phases of drug discovery and development. Mathematical models provide a useful tool to quantitatively characterize tumor growth dynamics and also optimize upcoming experiments. To the best of our knowledge, this is the first report where unperturbed growth of a large set of tumor cell lines (n = 28) has been systematically analyzed using a previously proposed model of nonlinear mixed effects (NLME). Exponential growth was identified as the governing mechanism in the majority of the cell lines, with constant rate values ranging from 0.0204 to 0.203 day-1 No common patterns could be observed across tumor types, highlighting the importance of combining information from different cell lines when evaluating drug activity. Overall, typical model parameters were precisely estimated using designs in which tumor size measurements were taken every 2 days. Moreover, reducing the number of measurements to twice per week, or even once per week for cell lines with low growth rates, showed little impact on parameter precision. However, a sample size of at least 50 mice is needed to accurately characterize parameter variability (i.e., relative S.E. values below 50%). This work illustrates the feasibility of systematically applying NLME models to characterize tumor growth in drug discovery and development, and constitutes a valuable source of data to optimize experimental designs by providing an a priori sampling window and minimizing the number of samples required.

Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange.

We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca(2+) concentration dynamics and other second-messenger signaling activities.

Structural templating of J-aggregates: Visualizing bis(monoacylglycero)phosphate domains in live cells.

Identifying the key structural and dynamical determinants that drive the association of biomolecules, whether in solution, or perhaps more importantly in a membrane environment, has critical implications for our understanding of cellular dynamics, processes, and signaling. With recent advances in high-resolution imaging techniques, from the development of new molecular labels to technical advances in imaging methodologies and platforms, researchers are now reaping the benefits of being able to directly characterize and quantify local dynamics, structures, and conformations in live cells and tissues. These capabilities are providing unique insights into association stoichiometries, interactions, and structures on sub-micron length scales. We previously examined the role of lipid headgroup chemistry and phase state in guiding the formation of pseudoisocyanine (PIC) dye J-aggregates on supported planar bilayers [Langmuir, 25, 10719]. We describe here how these same J-aggregates can report on the in situ formation of organellar membrane domains in live cells. Live cell hyperspectral confocal microscopy using GFP-conjugated GTPase markers of early (Rab5) and late (Rab7) endosomes revealed that the PIC J-aggregates were confined to domains on either the limiting membrane or intralumenal vesicles (ILV) of late endosomes, known to be enriched in the anionic lipid bis(monoacylglycero)phosphate (BMP). Correlated confocal fluorescence - atomic force microscopy performed on endosomal membrane-mimetic supported planar lipid bilayers confirmed BMP-specific templating of the PIC J-aggregates. These data provide strong evidence for the formation of BMP-rich lipid domains during multivesicular body formation and portend the application of structured dye aggregates as markers of cellular membrane domain structure, size, and formation.

Widely accessible method for superresolution fluorescence imaging of living systems.

Superresolution fluorescence microscopy overcomes the diffraction resolution barrier and allows the molecular intricacies of life to be revealed with greatly enhanced detail. However, many current superresolution techniques still face limitations and their implementation is typically associated with a steep learning curve. Patterned illumination-based superresolution techniques [e.g., stimulated emission depletion (STED), reversible optically-linear fluorescence transitions (RESOLFT), and saturated structured illumination microscopy (SSIM)] require specialized equipment, whereas single-molecule-based approaches [e.g., stochastic optical reconstruction microscopy (STORM), photo-activation localization microscopy (PALM), and fluorescence-PALM (F-PALM)] involve repetitive single-molecule localization, which requires its own set of expertise and is also temporally demanding. Here we present a superresolution fluorescence imaging method, photochromic stochastic optical fluctuation imaging (pcSOFI). In this method, irradiating a reversibly photoswitching fluorescent protein at an appropriate wavelength produces robust single-molecule intensity fluctuations, from which a superresolution picture can be extracted by a statistical analysis of the fluctuations in each pixel as a function of time, as previously demonstrated in SOFI. This method, which uses off-the-shelf equipment, genetically encodable labels, and simple and rapid data acquisition, is capable of providing two- to threefold-enhanced spatial resolution, significant background rejection, markedly improved contrast, and favorable temporal resolution in living cells. Furthermore, both 3D and multicolor imaging are readily achievable. Because of its ease of use and high performance, we anticipate that pcSOFI will prove an attractive approach for superresolution imaging.

Optimal sampling of antipsychotic medicines: a pharmacometric approach for clinical practice.

AIM: To determine optimal sampling strategies to allow the calculation of clinical pharmacokinetic parameters for selected antipsychotic medicines using a pharmacometric approach. METHODS: This study utilized previous population pharmacokinetic parameters of the antipsychotic medicines aripiprazole, clozapine, olanzapine, perphenazine, quetiapine, risperidone (including 9-OH risperidone) and ziprasidone. d-optimality was utilized to identify time points which accurately predicted the pharmacokinetic parameters (and expected error) of each drug at steady-state. A standard two stage population approach (STS) with MAP-Bayesian estimation was used to compare area under the concentration-time curves (AUC) generated from sparse optimal time points and rich extensive data. Monte Carlo Simulation (MCS) was used to simulate 1000 patients with population variability in pharmacokinetic parameters. Forward stepwise regression analysis was used to determine the most predictive time points of the AUC for each drug at steady-state. RESULTS: Three optimal sampling times were identified for each antipsychotic medicine. For aripiprazole, clozapine, olanzapine, perphenazine, risperidone, 9-OH risperidone, quetiapine and ziprasidone the CV% of the apparent clearance using optimal sampling strategies were 19.5, 8.6, 9.5, 13.5, 12.9, 10.0, 16.0 and 10.7, respectively. Using the MCS and linear regression approach to predict AUC, the recommended sampling windows were 16.5-17.5 h, 10-11 h, 23-24 h, 19-20 h, 16.5-17.5 h, 22.5-23.5 h, 5-6 h and 5.5-6.5 h, respectively. CONCLUSION: This analysis provides important sampling information for future population pharmacokinetic studies and clinical studies investigating the pharmacokinetics of antipsychotic medicines.

Differential expression and pharmacology of native P2X receptors in rat and primate sensory neurons.

Evidence suggesting the involvement of P2X2 and P2X3 in chronic pain has been obtained mostly from rodent models. Here we show that rodents may be poor predictors of P2X3 pharmacology in human. We demonstrate that monkey and human dorsal root ganglion (DRG) neurons do not express appreciable levels of P2X2 subunit, contrary to rat sensory neurons. Additionally, we report functional P2X3 activity in monkey DRG neurons and confirm the absence of functional P2X2/3 receptors. Interestingly, native P2X3 receptors in rat and monkey DRGs show similar agonist potency, but different antagonist potencies for TNP-ATP [2-O-(2,4,6-trinitrophenyl)-ATP] and RO51. This unexpected difference in antagonist potency was confirmed by comparing rat and human P2X3 receptors in HEK293 cells. Mutagenesis studies reveal that two extracellular residues, A197 and T202, are synergistically responsible for the potency drop in primate P2X3 receptors. These results uncover species-specific P2X3 pharmacology and identify key mechanisms impacting the translatability of potential analgesics targeting P2X3 receptors.

Control of P2X3 channel function by metabotropic P2Y2 utp receptors in primary sensory neurons.

Purinergic signaling contributes significantly to pain mechanisms, and the nociceptor-specific P2X3 ATP receptor channel is considered a target in pain therapeutics. Recent findings suggesting the coexpression of metabotropic P2Y receptors with P2X3 implies that ATP release triggers the activation of both ionotropic and metabotropic purinoceptors, with strong potential for functional interaction. Modulation of native P2X3 function by P2Y receptor activation was investigated in rat dorsal root ganglia (DRG) neurons using whole cell patch-clamp recordings. Application of the selective P2Y receptor agonist UTP decreased peak amplitudes of α,ß-meATP-evoked homomeric P2X3-mediated currents, but had no effect on heteromeric P2X2/3-mediated currents. Treatment with phospholipase C inhibitor U73122 significantly reversed P2X3 current inhibition induced by UTP-sensitive P2Y receptor activation. We previously reported the modulation of P2X receptors by phospholipids in DRG neurons and injection of exogenous phosphatidylinositol-4,5-bisphosphate (PIP(2)) fully reverses UTP-mediated regulation of P2X3 channel activity. Pharmacological as well as functional screening of P2Y receptor subtypes indicates the predominant involvement of P2Y2 receptor in P2X3 inhibition, and immunolocalization confirms a significant cellular coexpression of P2X3 and P2Y2 in rat DRG neurons. In summary, the function of P2X3 ATP receptor can be inhibited by P2Y2-mediated depletion of PIP(2). We propose that expression of P2Y2 purinoceptor in nociceptive sensory neurons provides an homeostatic mechanism to prevent excessive ATP signaling through P2X3 receptor channels.

Gasdermin D pores are dynamically regulated by local phosphoinositide circuitry.

Gasdermin D forms large, ~21 nm diameter pores in the plasma membrane to drive the cell death program pyroptosis. These pores are thought to be permanently open, and the resultant osmotic imbalance is thought to be highly damaging. Yet some cells mitigate and survive pore formation, suggesting an undiscovered layer of regulation over the function of these pores. However, no methods exist to directly reveal these mechanistic details. Here, we combine optogenetic tools, live cell fluorescence biosensing, and electrophysiology to demonstrate that gasdermin pores display phosphoinositide-dependent dynamics. We quantify repeated and fast opening-closing of these pores on the tens of seconds timescale, visualize the dynamic pore geometry, and identify the signaling that controls dynamic pore activity. The identification of this circuit allows pharmacological tuning of pyroptosis and control of inflammatory cytokine release by living cells.

Neuropathic Nav1.3-mediated sensitization to P2X activation is regulated by protein kinase C.

BACKGROUND: Increased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na+ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions. RESULTS: Using the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,ß-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,ß-meATP-induced Na+ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,ß-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,ß-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Nav1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Nav1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude. CONCLUSION: Neuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Nav1.3 subunit, and the function of the Nav1.3 channel is regulated by PKC.

Tyrosine phosphorylation of S1PR1 leads to chaperone BiP-mediated import to the endoplasmic reticulum.

Cell surface G protein-coupled receptors (GPCRs), upon agonist binding, undergo serine-threonine phosphorylation, leading to either receptor recycling or degradation. Here, we show a new fate of GPCRs, exemplified by ER retention of sphingosine-1-phosphate receptor 1 (S1PR1). We show that S1P phosphorylates S1PR1 on tyrosine residue Y143, which is associated with recruitment of activated BiP from the ER into the cytosol. BiP then interacts with endocytosed Y143-S1PR1 and delivers it into the ER. In contrast to WT-S1PR1, which is recycled and stabilizes the endothelial barrier, phosphomimicking S1PR1 (Y143D-S1PR1) is retained by BiP in the ER and increases cytosolic Ca2+ and disrupts barrier function. Intriguingly, a proinflammatory, but non-GPCR agonist, TNF-α, also triggered barrier-disruptive signaling by promoting S1PR1 phosphorylation on Y143 and its import into ER via BiP. BiP depletion restored Y143D-S1PR1 expression on the endothelial cell surface and rescued canonical receptor functions. Findings identify Y143-phosphorylated S1PR1 as a potential target for prevention of endothelial barrier breakdown under inflammatory conditions.

Cell-Matrix Interactions Regulate Functional Extracellular Vesicle Secretion from Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) are cell-secreted particles with broad potential to treat tissue injuries by delivering cargo to program target cells. However, improving the yield of functional EVs on a per cell basis remains challenging due to an incomplete understanding of how microenvironmental cues regulate EV secretion at the nanoscale. We show that mesenchymal stromal cells (MSCs) seeded on engineered hydrogels that mimic the elasticity of soft tissues with a lower integrin ligand density secrete ∼10-fold more EVs per cell than MSCs seeded on a rigid plastic substrate, without compromising their therapeutic activity or cargo to resolve acute lung injury in mice. Mechanistically, intracellular CD63+ multivesicular bodies (MVBs) transport faster within MSCs on softer hydrogels, leading to an increased frequency of MVB fusion with the plasma membrane to secrete more EVs. Actin-related protein 2/3 complex but not myosin-II limits MVB transport and EV secretion from MSCs on hydrogels. The results provide a rational basis for biomaterial design to improve EV secretion while maintaining their functionality.

Phase 1 trial of olaratumab monotherapy and in combination with chemotherapy in pediatric patients with relapsed/refractory solid and central nervous system tumors.

Olaratumab is a monoclonal antibody that specifically binds to platelet-derived growth factor receptor alpha (PDGFRα) and blocks receptor activation. We conducted a phase 1 trial to evaluate the safety of olaratumab and determine a recommended dose in combination with three different chemotherapy regimens in children. Patients <18 years with relapsed/refractory solid or central nervous system tumors were enrolled to two dose levels of olaratumab. Patients received olaratumab monotherapy at 15 mg/kg (Part A) or 20 mg/kg (Part B) on Days 1 and 8 of the first 21-day cycle, followed by olaratumab combined with standard fixed doses of chemotherapy with doxorubicin, vincristine/irinotecan, or high-dose ifosfamide by investigator choice for subsequent 21-day cycles. In Part C, patients received olaratumab 20 mg/kg plus assigned chemotherapy for all cycles. Parts A-C enrolled 68 patients across three chemotherapy treatment arms; olaratumab in combination with doxorubicin (N = 16), vincristine/irinotecan (N = 26), or ifosfamide (N = 26). Three dose-limiting toxicities (DLTs) occurred during olaratumab monotherapy (at 15 mg/kg, grade [G] 4 alanine aminotransferase [ALT]; at 20 mg/kg, G3 lung infection and G3 gamma-glutamyl transferase). One DLT occurred during vincristine/irinotecan with olaratumab 20 mg/kg therapy (G3 ALT). Treatment-emergent adverse events ≥G3 in >25% of patients included neutropenia, anemia, leukopenia, lymphopenia, and thrombocytopenia. Pharmacokinetic profiles of olaratumab with chemotherapy were within the projected range based on adult data. There was one complete response (rhabdomyosarcoma [Part B vincristine/irinotecan arm]) and three partial responses (two rhabdomyosarcoma [Part A doxorubicin arm and Part C doxorubicin arm]; one pineoblastoma [Part B vincristine/irinotecan arm]). Olaratumab was tolerable and safely administered in combination with chemotherapy regimens commonly used in children and adolescents.

Circulating Tumor Cells and Biomarker Modulation with Olaratumab Monotherapy Followed by Olaratumab plus Doxorubicin: Phase Ib Study in Patients with Soft-Tissue Sarcoma.

This phase Ib study enumerated whole blood circulating tumor cells (CTC) and evaluated biomarkers in patients with potentially resectable soft-tissue sarcoma (STS) treated with olaratumab monotherapy (20 mg/kg) for one cycle followed by up to six cycles of olaratumab (20 mg/kg, cycles 1-2; 15 mg/kg, cycles 3-7) plus doxorubicin (75 mg/m2 on day 1). CTCs, platelet-derived growth factor receptors (PDGFR), and PDGF ligand expression in tumor tissue pre- and post-olaratumab monotherapy were evaluated. Antitumor activity, safety, pharmacokinetics, and PET/biomarker association with clinical outcome were assessed. Of 51 treated patients, 35, 43, and 37 were evaluable for CTC enumeration, PDGFRs, and PDGF ligand expression, respectively. An increase in CTCs at cycle 1 day 8 was observed, followed by a significant reduction by cycle 3 day 1 or 30-day follow-up. Decrease in CTC counts after olaratumab monotherapy was higher in patients with disease control than without disease control (57.9% vs. 31.2%). Baseline IHC expression was positive in most patients for PDGFRα [n = 31 (72.1%)] and PDGFRß [n = 36 (83.7%)]. Similar rates were observed post-olaratumab monotherapy [PDGFRα, n = 30 (69.8%); PDGFRß, n = 33 (76.7%)]. Eleven patients (29.7%) showed a 30% reduction by RT-PCR in PDGFRα at cycle 2. PDGFR expression and PET response showed no correlation with clinical outcome. Safety and pharmacokinetic profiles were consistent with previous reports. This study, the first to use a validated method for CTC detection, confirms that CTC enumeration in STS is feasible. However, no correlation was observed between PDGFRα expression and clinical outcome.

A rationally enhanced red fluorescent protein expands the utility of FRET biosensors.

Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools to illuminate spatiotemporal regulation of cell signaling in living cells, but the utility of the red spectrum for biosensing was limited due to a lack of bright and stable red fluorescent proteins. Here, we rationally improve the photophysical characteristics of the coral-derived fluorescent protein TagRFP-T. We show that a new single-residue mutant, super-TagRFP (stagRFP) has nearly twice the molecular brightness of TagRFP-T and negligible photoactivation. stagRFP facilitates significant improvements on multiple green-red biosensors as a FRET acceptor and is an efficient FRET donor that supports red/far-red FRET biosensing. Capitalizing on the ability of stagRFP to couple with multiple FRET partners, we develop a novel multiplex method to examine the confluence of signaling activities from three kinases simultaneously in single living cells, providing evidence for a role of Src family kinases in regulating growth factor induced Akt and ERK activities.

Pharmacokinetics of doxorubicin following concomitant intravenous administration of olaratumab (IMC-3G3) to patients with advanced soft tissue sarcoma.

BACKGROUND: Olaratumab, a fully human monoclonal antibody, selectively binds to human platelet-derived growth factor receptor alpha and blocks ligand binding. This study assessed the effect of olaratumab on the pharmacokinetics (PK) of doxorubicin and the safety of olaratumab alone and in combination with doxorubicin. METHODS: This open-label randomized phase 1 trial enrolled 49 patients ages 27 to 83 with metastatic or locally advanced soft tissue sarcoma (STS). Patients participated in 21-day treatment cycles (up to 8) until they met discontinuation criteria. In cycles 1 and 2, patients received olaratumab (15 mg/kg in Part A, 20 mg/kg in Part B) and doxorubicin (75 mg/m2 ). In cycles 3 through 8, patients continued combination treatment (15 mg/kg olaratumab + doxorubicin). Effect of olaratumab on PK of doxorubicin was determined in patients who received all doses in cycles 1 and 2. RESULTS: PK properties of doxorubicin administered alone or in combination with olaratumab (15 or 20 mg/kg) were similar for AUC(0-tlast ), AUC(0-∞), and Cmax . PK properties of olaratumab (15 or 20 mg/kg) were also similar when administered alone or in combination with doxorubicin. Three patients died (2 of disease progression and 1 of neutropenic enterocolitis). Fatigue and nausea (>75% of patients) were the most common treatment-emergent adverse events (TEAEs). Other common TEAEs included musculoskeletal pain, mucositis, constipation, and diarrhea. CONCLUSIONS: Olaratumab at 15 or 20 mg/kg before doxorubicin infusion had no clinically relevant effect on systemic exposure to doxorubicin compared with doxorubicin alone in patients with metastatic or locally advanced STS.

EphB1 interaction with caveolin-1 in endothelial cells modulates caveolae biogenesis.

Caveolae, the cave-like structures abundant in endothelial cells (ECs), are important for multiple signaling processes such as production of nitric oxide and caveolae-mediated intracellular trafficking. Using superresolution microscopy, fluorescence resonance energy transfer, and biochemical analysis, we observed that the EphB1 receptor tyrosine kinase constitutively interacts with caveolin-1 (Cav-1), the key structural protein of caveolae. Activation of EphB1 with its ligand Ephrin B1 induced EphB1 phosphorylation and the uncoupling EphB1 from Cav-1 and thereby promoted phosphorylation of Cav-1 by Src. Deletion of Cav-1 scaffold domain binding (CSD) motif in EphB1 prevented EphB1 binding to Cav-1 as well as Src-dependent Cav-1 phosphorylation, indicating the importance of CSD in the interaction. We also observed that Cav-1 protein expression and caveolae numbers were markedly reduced in ECs from EphB1-deficient (EphB1-/-) mice. The loss of EphB1 binding to Cav-1 promoted Cav-1 ubiquitination and degradation, and hence the loss of Cav-1 was responsible for reducing the caveolae numbers. These studies identify the crucial role of EphB1/Cav-1 interaction in the biogenesis of caveolae and in coordinating the signaling function of Cav-1 in ECs.