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Figure S1: Impedance barrier function assay: The effect of S1P and Ex82 was analyzed using a transendothelial electrical impedance assay. S1P treatment (10 nM) of an endothelial monolayer strongly increases electrical impedance (B) vs control (A) whereas Ex82 has the opposite effect and significantly decreases electrical impedance (D). These results are consistent with the known barrier function of S1P1. In addition, pretreatment with Ex82 blocked the S1P dependent increase in electrical impedance (C).

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Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

Supplemental Table 1: Activities of LY2510924.acetate in ligand binding assays of selected chemokine receptors and other GPCRs

LY573636-sodium (tasisulam) is a small molecule antitumor agent with a novel mechanism of action currently being investigated in a variety of human cancers. In vitro, tasisulam induced apoptosis via the intrinsic pathway, resulting in cytochrome c release and caspase-dependent cell death. Using high content cellular imaging and subpopulation analysis of a wide range of in vitro and in vivo cancer models, tasisulam increased the proportion of cells with 4N DNA content and phospho-histone H3 expression, leading to G2–M accumulation and subsequent apoptosis. Tasisulam also blocked VEGF, epidermal growth factor, and fibroblast growth factor–induced endothelial cell cord formation but did not block acute growth factor receptor signaling (unlike sunitinib, which blocks VEGF-driven angiogenesis at the receptor kinase level) or induce apoptosis in primary endothelial cells. Importantly, in vivo phenocopying of in vitro effects were observed in multiple human tumor xenografts. Tasisulam was as effective as sunitinib at inhibiting neovascularization in a Matrigel plug angiogenesis assay in vivo and also caused reversible, non G2–M–dependent growth arrest in primary endothelial cells. Tasisulam also induced vascular normalization in vivo. Interestingly, the combination of tasisulam and sunitinib significantly delayed growth of the Caki-1 renal cell carcinoma model, whereas neither agent was active alone. These data show that tasisulam has a unique, dual-faceted mechanism of action involving mitotic catastrophe and antiangiogenesis, a phenotype distinct from conventional chemotherapies and published anticancer agents. Mol Cancer Ther; 10(11); 2168–78. ©2011 AACR.

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Figure S2: Co-targeting S1P1 and VEGFR2 pathways induced reduction in permeability of retinal vessels. Ter 119 staining (panels A) visualize red blood cells and show that the combination of DC101 and the S1P1 antagonist in the retina led to reduction of the areas of hemorrhage within the vascular plexus and restricted only to the angiogenic front indicating that the remaining vessels in the remodeled area are less permeable.

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These figures contain extensions of data that would not fit in the primary manuscript but are important for a complete understanding of the work. These include analysis of other data sets, correlations between genomic profiling our stromal subtyping and information on bioinformatics and animal models.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573–86. ©2016 AACR.

Supplementary Tables S1-S3, Figures S1-S4

Purpose:Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The EWS/ETS fusion transcription factor that drives ES pathobiology was previously demonstrated to modulate cyclin D1 expression. In this study, we evaluated abemaciclib, a small-molecule CDK4 and CDK6 (CDK4 and 6) inhibitor currently under clinical investigation in pediatric solid tumors, in preclinical models of ES.Experimental Design:Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the in vitro response of ES cell lines to abemaciclib. We then evaluated abemaciclib in vivo in cell line–derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of ES as either a monotherapy or in combination with chemotherapy.Results:Abemaciclib induced quiescence in ES cell lines via a G1 cell-cycle block, characterized by decreased proliferation and reduction of Ki-67 and FOXM1 expression and retinoblastoma protein (RB) phosphorylation. In addition, abemaciclib reduced DNMT1 expression and promoted an inflammatory immune response as measured by cytokine secretion, antigen presentation, and interferon pathway upregulation. Single-agent abemaciclib reduced ES tumor volume in preclinical mouse models and, when given in combination with doxorubicin or temozolomide plus irinotecan, durable disease control was observed.Conclusions:Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.

These figures contain extensions of data that would not fit in the primary manuscript but are important for a complete understanding of the work. These include analysis of other data sets, correlations between genomic profiling our stromal subtyping and information on bioinformatics and animal models.

This file contains details too lengthy to put in the primary manuscript file as well as methods for supplemental data

Supplemental methodology, specifically the in vitro cord formation assay and the enzyme-linked immunosorbent assay.

These are the figure legends for the supplemental figures and tables.

Purpose: Checkpoint kinase 1 (CHK1) is a key regulator of the DNA damage response and a mediator of replication stress through modulation of replication fork licensing and activation of S and G2–M cell-cycle checkpoints. We evaluated prexasertib (LY2606368), a small-molecule CHK1 inhibitor currently in clinical testing, in multiple preclinical models of pediatric cancer. Following an initial assessment of prexasertib activity, this study focused on the preclinical models of neuroblastoma.Experimental Design: We evaluated the antiproliferative activity of prexasertib in a panel of cancer cell lines; neuroblastoma cell lines were among the most sensitive. Subsequent Western blot and immunofluorescence analyses measured DNA damage and DNA repair protein activation. Prexasertib was investigated in several cell line–derived xenograft mouse models of neuroblastoma.Results: Within 24 hours, single-agent prexasertib promoted γH2AX–positive double-strand DNA breaks and phosphorylation of DNA damage sensors ATM and DNA–PKcs, leading to neuroblastoma cell death. Knockdown of CHK1 and/or CHK2 by siRNA verified that the double-strand DNA breaks and cell death elicited by prexasertib were due to specific CHK1 inhibition. Neuroblastoma xenografts rapidly regressed following prexasertib administration, independent of starting tumor volume. Decreased Ki67 and increased immunostaining of endothelial and pericyte markers were observed in xenografts after only 6 days of exposure to prexasertib, potentially indicating a swift reduction in tumor volume and/or a direct effect on tumor vasculature.Conclusions: Overall, these data demonstrate that prexasertib is a specific inhibitor of CHK1 in neuroblastoma and leads to DNA damage and cell death in preclinical models of this devastating pediatric malignancy. Clin Cancer Res; 23(15); 4354–63. ©2017 AACR.

Objective To provide guidance to providers on how to improve the patient experience for women with recurrent urinary tract infections (rUTIs). Methods The recently updated 2019 American Urological Association (AUA) guidelines for recurrent uncomplicated acute cystitis and other contemporary publications pertaining to recurrent UTIs in women were reviewed. These data were used to summarize practice-based methodology to formulate recommendations with an emphasis on education to enhance the patient experience. Results We summarize the guideline-based management of patients with rUTI and augment this with a patient-focused perspective to provide recommendations for how to best counsel patients regarding evaluation, treatment using antibiotic stewardship practices, and a comprehensive prevention plan. We focus on patient education as the foundation for successful provider-patient relationships as well as patient compliance with care pathways. Conclusion rUTIs are costly, time-consuming, and painful for patients. For providers, rUTIs represent a frustrating aspect of clinical care facing the balance of antibiotic stewardship with effective treatment and patient expectations with limited data supporting nonantibiotic therapies. Urologists have the skills and knowledge to provide this patient population with competent and compassionate care. By investing in these patients, being responsive to their concerns and offering education, patients will have a better overall experience with this chronic condition.

OBJECTIVE: To prospectively evaluate the effectiveness of OnabotulinumtoxinA (BTX-A) on neurogenic overactive bladder (nOAB) in adults with congenital spinal dysraphism (CSD). SUBJECTS AND METHODS: We conducted a prospective, nonrandomized pilot study of 24 adults with CSD and nOAB. Patients were evaluated with baseline video-urodynamics (UDS) and validated questionnaires, underwent injection 200U BTX-A, and then underwent repeat evaluation with questionnaires and UDS 1–3 months post injection. A high-risk subgroup was separately analyzed based on adverse clinical characteristics (i.e. decrease bladder compliance, vesicoureteral reflux, hydronephrosis, chronic kidney disease). RESULTS: BTX-A injection improved patient recorded outcome measures seen in both I-QOL Score total (67.9 vs 75.5, p= 0.007) and Neurogenic Bladder Symptom Score total (38.0 vs 29.0, p=0.001). On UDS, BTX-A injection significantly improved end filling pressure (16.0 vs 8.8, p= 0.036) and also improved bladder compliance (mL/cm H2O) (89.38 vs 135.81, p= 0.445). High-risk patients were found to have similar improvements in most subjective questionnaire scoring, a significant decrease in end filling pressures, and improved bladder compliance on UDS. CONCLUSION: BTX-A can be used as an effective treatment in adults with CSD. We found that BTX-A significantly improved quality of life from patient reported outcome measurements as well as improving end filling pressures and bladder compliance. These improvements were seen even within our high-risk subgroup. Further studies are needed to evaluate long-term efficacy and appropriate follow-up of this at-risk population.

INTRODUCTION AND OBJECTIVE:OnabotulinumtoxinA (BTX-A) injection is a well-established management strategy in the treatment of patients with neurogenic overactive bladder (NOAB). Few studies, howeve...

Purpose: Ewing sarcoma (ES) is a rare and highly malignant cancer that occurs in the bone and surrounding tissue of children and adolescents. The EWS/ETS fusion transcription factor that drives ES pathobiology was previously demonstrated to modulate cyclin D1 expression. In this study, we evaluated abemaciclib, a small-molecule CDK4 and CDK6 (CDK4 and 6) inhibitor currently under clinical investigation in pediatric solid tumors, in preclinical models of ES. Experimental Design: Using Western blot, high-content imaging, flow cytometry, ELISA, RNA sequencing, and CpG methylation assays, we characterized the in vitro response of ES cell lines to abemaciclib. We then evaluated abemaciclib in vivo in cell line–derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models of ES as either a monotherapy or in combination with chemotherapy. Results: Abemaciclib induced quiescence in ES cell lines via a G1 cell-cycle block, characterized by decreased proliferation and reduction of Ki-67 and FOXM1 expression and retinoblastoma protein (RB) phosphorylation. In addition, abemaciclib reduced DNMT1 expression and promoted an inflammatory immune response as measured by cytokine secretion, antigen presentation, and interferon pathway upregulation. Single-agent abemaciclib reduced ES tumor volume in preclinical mouse models and, when given in combination with doxorubicin or temozolomide plus irinotecan, durable disease control was observed. Conclusions: Collectively, our data demonstrate that the antitumor effects of abemaciclib in preclinical ES models are multifaceted and include cell-cycle inhibition, DNA demethylation, and immunogenic changes.

OBJECTIVE This retrospective study describes procedures of choice in management of patients with primary prolapse compared with recurrence prolapse patients by fellowship-trained surgeons. METHODS Surgically managed primary and recurrent prolapse cases from 2012 to 2015 at Houston Methodist Hospital were reviewed. Baseline characteristics, compartment defects, and stage were compared. Mean interval from the index surgeries to management of prolapse recurrence was recorded. In recurrence cases, mesh complaints were noted if present. Primary outcome was the procedure type used to manage cases of recurrence and primary prolapse. Logistic regression was used to determine odds ratio (OR) for the procedure of choice in recurrence and primary repairs of prolapse. RESULTS Of 386 cases reviewed, 379 met criteria for inclusion; 25.8% of repairs were for recurrence. Recurrence patients were significantly older than primary cases (mean, 63.6 vs 60.5; P = 0.03) and had been postmenopausal for longer (P = 0.004). Median time interval to surgical management of recurrence was 8 years. Thirty percent of recurrence patients treated previously by mesh had mesh complaints. There was no difference in the distribution of defects or stage. Sacrocolpopexy was more frequently used to manage recurrent prolapse (OR, 2.6334; P < 0.0005). Vaginal mesh repairs showed no difference in utilization. Uterosacral ligament fixation (OR, 0.347; P = 0.002) was used more often in primary prolapse. Anterior colporrhaphy (OR, 0.398; P = 0.0005) and uterosacral ligament fixation (OR, 0.347; P = 0.002) were performed less in recurrence cases. CONCLUSION Fellowship-trained urogynecologists at this institution utilize sacrocolpopexy mesh more frequently in recurrent prolapse, and uterosacral ligament fixation was used more frequently in primary prolapse cases.

Merestinib is an oral multi-kinase inhibitor targeting a limited number of oncokinases including MET, AXL, RON and MKNK1/2. Here, we report that merestinib inhibits neurotrophic receptor tyrosine kinases NTRK1/2/3 which are oncogenic drivers in tumors bearing NTRK fusion resulting from chromosomal rearrangements. Merestinib is shown to be a type II NTRK1 kinase inhibitor as determined by x-ray crystallography. In KM-12 cells harboring TPM3-NTRK1 fusion, merestinib exhibits potent p-NTRK1 inhibition in vitro by western blot and elicits an anti-proliferative response in two- and three-dimensional growth. Merestinib treatment demonstrated profound tumor growth inhibition in in vivo cancer models harboring either a TPM3-NTRK1 or an ETV6-NTRK3 gene fusion. To recapitulate resistance observed from type I NTRK kinase inhibitors entrectinib and larotrectinib, we generated NIH-3T3 cells exogenously expressing TPM3-NTRK1 wild-type, or acquired mutations G595R and G667C in vitro and in vivo. Merestinib blocks tumor growth of both wild-type and mutant G667C TPM3-NTRK1 expressing NIH-3T3 cell-derived tumors. These preclinical data support the clinical evaluation of merestinib, a type II NTRK kinase inhibitor (NCT02920996), both in treatment naïve patients and in patients progressed on type I NTRK kinase inhibitors with acquired secondary G667C mutation in NTRK fusion bearing tumors.

INTRODUCTION Chronic antithrombotic therapy is common among patients requiring surgery for pelvic organ prolapse because of age and comorbidities. The impact of chronic anticoagulation on postoperative complications in pelvic organ prolapse surgery has not been investigated. This study aims to determine if patients on chronic antithrombotic therapy are at increased risk for postoperative complications. METHODS This retrospective cohort study included women having prolapse surgery from 2012 to 2015, identified by Current Procedural Terminology codes, excluding patients undergoing concomitant major nonurogynecologic procedures. Baseline characteristics were compared and all procedures performed, operative duration, estimated blood loss, and length of hospitalization. Complications (blood transfusion, intensive care unit admission, reoperation, readmission, hematoma, thromboembolic event, and infection) were compared in women on chronic antithrombotic therapy and controls. Logistic regression determined odds ratio (OR) for complications in patients on chronic antithrombotics. Complications were graded by the Clavien-Dindo classification. RESULTS A total of 388 charts were reviewed, and 386 patients met inclusion criteria. Twenty-one of the 386 patients were on chronic antithrombotic therapy. Chronic antithrombotic therapy increased overall complications (OR, 6.8; P < 0.0005), blood transfusion (OR, 165; P < 0.001), intensive care unit admission (OR, 19.10; P < 0.004), hospital readmission (OR, 20.7; P < 0.0005), vaginal hematoma (OR, 554.1; P < 0.001), infection (OR, 22.44; P < 0.004), and complications that required specific additional follow-up (OR, 9.42; P < 0.0005). There were no thromboembolic events. Antithrombotic therapy did not significantly increase reoperation rates (OR, 3.8; P = 0.275). Findings were maintained when adjusting for covariates of age and body mass index. CONCLUSIONS Postoperative surgical complications after prolapse repair procedures are increased in patients who use chronic antithrombotic medication, the majority of cases are successfully managed conservatively.

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573–86. ©2016 AACR.

Inhibition of VEGFR signaling is an effective treatment for renal cell carcinoma, but resistance continues to be a major problem. Recently, the sphingosine phosphate (S1P) signaling pathway has been implicated in tumor growth, angiogenesis, and resistance to antiangiogenic therapy. S1P is a bioactive lipid that serves an essential role in developmental and pathologic angiogenesis via activation of the S1P receptor 1 (S1P1). S1P1 signaling counteracts VEGF signaling and is required for vascular stabilization. We used in vivo and in vitro angiogenesis models including a postnatal retinal angiogenesis model and a renal cell carcinoma murine tumor model to test whether simultaneous inhibition of S1P1 and VEGF leads to improved angiogenic inhibition. Here, we show that inhibition of S1P signaling reduces the endothelial cell barrier and leads to excessive angiogenic sprouting. Simultaneous inhibition of S1P and VEGF signaling further disrupts the tumor vascular beds, decreases tumor volume, and increases tumor cell death compared with monotherapies. These studies suggest that inhibition of angiogenesis at two stages of the multistep process may maximize the effects of antiangiogenic therapy. Together, these data suggest that combination of S1P1 and VEGFR-targeted therapy may be a useful therapeutic strategy for the treatment of renal cell carcinoma and other tumor types.

The purpose is to present the first report and describe our novel technique for intracorporeal continent cutaneous diversion after robotic cystectomy. After completion of robot-assisted cystectomy using a standard six-port transperitoneal technique, three additional ports are placed, and the robot is redocked laterally over the patient's right side in the modified lateral position. Our technique replicates step-by-step the principles of the open approach. Ileocolonic anastomosis, ureteroenteral anastomoses, and construction of a hand-sewn right colonic pouch are all performed intracorporeally. Tapering of efferent ileal limb and reinforcement of the ileocecal valve are performed via the extraction site, while the stoma is matured through a prospective port site. Successful robotic intracorporeal creation of a modified Indiana pouch was achieved. Operative time for diversion was 3 hours, with negligible blood loss, and without any intraoperative complications. No major (Clavien III–V) 90-day complications were observed. At a follow-up of 1 year, the patient continues to catheterize without difficulty. We demonstrate the first description of robotic intracorporeal continent cutaneous urinary diversion after robot-assisted cystectomy. We present a systematic minimally invasive approach, replicating the principles of open surgery, which is technically feasible and safe with a good functional result.

Immigration is currently a combustible social issue in the United States, contributing to national political polarization. The Minuteman Project is one prominent group shaping contemporary immigration politics. This study explores the Utah chapter of this movement and explains why people join. In social movement terms, we highlight which grievances—material, cultural, or value-oriented—predict movement recruitment. This research incorporates a range of qualitative data: in-depth interviews, primary documents, and observation. Our fundamental finding is that contrary to a dominant belief, material interests are not driving anti-immigrant activism. Instead, we find that this activism grows out of the intersection of value-oriented and cultural grievances. Integrating four bodies of sociological theory, we find that values and culture—more than material interests—motivate people to become members of this important social movement.

This study explores the disaster response, political transformation and community development prospects of the Philippine community of Guinsaugon, a village devastated by a 2006 landslide. Utilizing a social justice perspective, we analyze a range of qualitative data collected over a 5-year period to understand how linking capital functions following a major social disruption. An understudied form of social capital and linking capital features embeddedness, shared values and mutual goals between individuals and groups that are divided by cleavages of class, power and privilege. We uncovered three major outcomes. First, an existing village institution—Cristo Rey High School—was a principal agent of distributive justice, providing immediate material goods and social support to the survivors. This local institution was deeply embedded in the village, yet maintained extensive ties with outside groups that provided crucial resources. Second, linking capital contributed to procedural justice and political transformation. The disaster sparked many emigrants to return to the village and form The ATHena Project: Advocacy for Transparency and Honesty, a civil society organization that promoted accountability and helped dethrone a local political dynasty. Finally, our study uncovered a crucial limitation of linking capital. This powerful social resource did not help fulfill essential elements of long-term community development, such as helping secure land, sustainable jobs or infrastructural development for re-located Guinsaugon villagers. In this way, we underscore the importance of a strong developmental state in post-disaster recovery. Without it, some of the gains derived from linking capital do not endure, undermining the advances of distributive and procedural justice that followed a major social disruption.