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STAT3, IL-6 and inflammatory response pathway signaling associated gene expression is upregulated in Group A EPN.

PDF file 98K, Microscopy data with representative photos and quantification

Experimentally determined STAT3 upregulated genes.

PDF file 31K, Autophagic flux by flow cytometry in vemurafenib treated cells

Ependymoma (EPN) in childhood is a brain tumor with substantial mortality. Inflammatory response has been identified as a molecular signature of high-risk Group A EPN. To better understand the biology of this phenotype and aid therapeutic development, transcriptomic data from Group A and B EPN patient tumor samples, and additional malignant and normal brain data, were analyzed to identify the mechanism underlying EPN Group A inflammation. Enrichment of IL6 and STAT3 pathway genes were found to distinguish Group A EPN from Group B EPN and other brain tumors, implicating an IL6 activation of STAT3 mechanism. EPN tumor cell growth was shown to be dependent on STAT3 activity, as demonstrated using shRNA knockdown and pharmacologic inhibition of STAT3 that blocked proliferation and induced apoptosis. The inflammatory factors secreted by EPN tumor cells were shown to reprogram myeloid cells, and this paracrine effect was characterized by a significant increase in pSTAT3 and IL8 secretion. Myeloid polarization was shown to be dependent on tumor secretion of IL6, and these effects could be reversed using IL6-neutralizing antibody or IL6 receptor–targeted therapeutic antibody tocilizumab. Polarized myeloid cell production of IL8 drove unpolarized myeloid cells to upregulate CD163 and to produce a number of proinflammatory cytokines. Collectively, these findings indicate that constitutive IL6/STAT3 pathway activation is important in driving tumor growth and inflammatory cross-talk with myeloid cells within the Group A EPN microenvironment. Effective design of Group A–targeted therapy for children with EPN may require reversal of this potentially immunosuppressive and protumor pathway. Cancer Immunol Res; 3(10); 1165–74. ©2015 AACR.

FDA panel: Details of drug screen library of 97 FDA approved anticancer active compounds. FDA drug targets: Genes corresponding to known protein targets of compounds in FDA approved oncology drug screen library, corresponding Affymetrix probeset IDs and literature source. GSEA of axitinib Tx: Axitinib-induced enrichment and depletion of MSigDB "Hallmark" genesets in transcriptomic profiles of axitinib-treated versus untreated controls.

IL-6 is the driving cytokine for immune suppressive phenotype observed in 811 CM monocytes.

S3I-201 effects on cell cycle EPN cell line 811 and apoptosis in EPN cell line 723.

PDF file 186K, Figures legends for supplementary data figures

Children with ependymoma (EPN) are cured in less than 50% of cases, with little improvement in outcome over the last several decades. Chemotherapy has not affected survival in EPN, due in part to a lack of preclinical models that has precluded comprehensive drug testing. We recently developed two human EPN cell lines harboring high-risk phenotypes which provided us with an opportunity to execute translational studies. EPN and other pediatric brain tumor cell lines were subject to a large-scale comparative drug screen of FDA-approved oncology drugs for rapid clinical application. The results of this in vitro study were combined with in silico prediction of drug sensitivity to identify EPN-selective compounds, which were validated by dose curve and time course modeling. Mechanisms of EPN-selective antitumor effect were further investigated using transcriptome and proteome analyses. We identified three classes of oncology drugs that showed EPN-selective antitumor effect, namely, (i) fluorinated pyrimidines (5-fluorouracil, carmofur, and floxuridine), (ii) retinoids (bexarotene, tretinoin and isotretinoin), and (iii) a subset of small-molecule multireceptor tyrosine kinase inhibitors (axitinib, imatinib, and pazopanib). Axitinib's antitumor mechanism in EPN cell lines involved inhibition of PDGFRα and PDGFRβ and was associated with reduced mitosis-related gene expression and cellular senescence. The clinically available, EPN-selective oncology drugs identified by our study have the potential to critically inform design of upcoming clinical studies in EPN, in particular for those children with recurrent EPN who are in the greatest need of novel therapeutic approaches. Mol Cancer Ther; 17(9); 1984–94. ©2018 AACR.

PDF file 121K, Incucyte growth analysis and western blot analysis

Apoptosis time courses for EPN cell lines 811 and 928 treated with floxuridine, bexarotene, and axitinib all at 1µM, as measured by live cell imaging (IncuCyte) ofcaspase 3/7 activation (green objects/mm2). Idarubicin (1µM) provides a positive control for apoptosis and untreated cells serve as a negative control.

Ratio of Group A EPN IL6/STAT3 pathway genes in tumor versus the myeloid compartments.

Consensus NMF of 81 primary ependymoma samples at k = 3 delineates two posterior fossa subgroups in the study cohort.

Supplemental Figure Legends

PDF file 2464K, Timeline of treatments and clinical responses

PDF file 117K,Propidium idodide results and western blot analysis

PDF file 17K, Growth curve with vemurafenib treatments

Expression of EPN-selective drug targets in primary PFA patient samples versus cerebellum. Gene expression for (A) RARB, (B) PDGFRB, (C) VEGFR2 (KDR) and (D) VEGFC in twelve PFA patient samples and 4 pediatric cerebellum samples. Red bars represent mean and standard deviation. P-values = student's t-test.

Astrocytomas account for the majority of malignant brain tumors diagnosed in both adult and pediatric patients. The therapies available to treat these neoplasms are limited, and the prognosis associated with high-grade lesions is extremely poor. Mer (MerTK) and Axl receptor tyrosine kinases (RTK) are expressed at abnormally high levels in a variety of malignancies, and these receptors are known to activate strong antiapoptotic signaling pathways that promote oncogenesis. In this study, we found that Mer and Axl mRNA transcript and protein expression were elevated in astrocytic patient samples and cell lines. shRNA-mediated knockdown of Mer and Axl RTK expression led to an increase in apoptosis in astrocytoma cells. Apoptotic signaling pathways including Akt and extracellular signal–regulated kinase 1/2, which have been shown to be activated in resistant astrocytomas, were downregulated with Mer and Axl inhibition whereas poly(ADP-ribose) polymerase cleavage was increased. Furthermore, Mer and Axl shRNA knockdown led to a profound decrease of astrocytoma cell proliferation in soft agar and a significant increase in chemosensitivity in response to temozolomide, carboplatin, and vincristine treatment. Our results suggest Mer and Axl RTK inhibition as a novel method to improve apoptotic response and chemosensitivity in astrocytoma and provide support for these oncogenes as attractive biological targets for astrocytoma drug development. Mol Cancer Ther; 9(5); 1298–307. ©2010 AACR.

PDF file - 1806K, Supplementary Figure S1. Hierarchical clustering of breast cancer cell lines transduced with an autophagy-focused shRNA library produced two main clusters. Supplementary Figure S2. ATG5, ATG7 and BECN1 shRNAs decreased protein levels in all cell lines tested. Supplementary Figure S3. Autophagy inhibition induces cell death in some cell lines but not others. MCF7, MDAMB231 and MDAMB468 cells were transduced with a lentivirus expressing shRNAs against ATG7 or BECN1, plated and evaluated for cell death with propidium iodide (red) staining after 48 h. Supplementary Figure S4. Triple negative cell lines have constitutively activated JAK/STAT pathway and autophagy regulates their STAT3 phosphorylation status.

MOV file - 3729K, MDAMB231 cells were transduced with a Non-silencing shRNA and imaged for 48h.

MOV file - 3141K, MCF7 cells were transduced with a Non-silencing shRNA and imaged for 48h.

PDF file - 117K, Supplementary table 1. Changes in shRNAs included in the autophagy library per cell line. Cells were transduced with a shRNA library containing autophagy-focused shRNAs. After transduction, the first sample (Post-transduction, PT) was collected. Growth sample (G) was collected after 15 days in culture. Both samples were done in quintuplicate. Table shows comparison of PT versus G samples. shRNAs were considered to be up (Sign Tag = U), or down-regulated (Sign Tag = D) in the growth sample when ANOVA p-value < 0.05 and fold change absolute value > 2.

MOV file - 2644K, MDAMB231 cells were transduced with an ATG7 shRNA and imaged for 48h.

MOV file - 2592K, MDAMB468 cells were transduced with an ATG7 shRNA and imaged for 48h.

MOV file - 2566K, MCF7 cells were transduced with an ATG7 shRNA and imaged for 48h.

Louisiana rice is attacked by a complex of lepidopteran stem borers that injure the vegetative and reproductive stages of rice, reducing tillering and grain output. Currently, insecticidal seed treatments are used in Louisiana as a preemptive means to control stem borers in rice. Our objective was to better understand the spatial distribution of stem borers in Louisiana rice fields. Thirteen nontreated commercial rice fields in Louisiana were mapped using GPS software and surveyed for stem borer damage in 2017 and 2018. Damage was recorded by counting the number of blank panicles (whiteheads) within quadrats located at different areas in each field. An ANOVA, spatial interpolation, hotspot analysis, and negative binomial regression were used to determine where stem borer injury was concentrated and whether the distribution of these pests was influenced by rice stand count, field composition, and site location. The results show that stem borers are typically aggregated along field edges, with population density decreasing toward the center of rice fields. Stem borer damage was 4.2- and 3.9-fold greater along field edges than in field centers in 2017 and 2018, respectively. Hotspot spatial analysis revealed 41.7 and 52.1% of low-density clusters occurred at or near field centers, respectively. Negative binomial regression revealed stem borer injury declines with increasing distance from field edges, with regional differences in infestation levels. This suggests stem borer control efforts could be spatially targeted to improve the efficiency of pest management.