Validation of a non-oncogene encoded vulnerability to exportin 1 inhibition in pediatric renal tumors.

BACKGROUND: Malignant rhabdoid tumors (MRTs) and Wilms' tumors (WTs) are rare and aggressive renal tumors of infants and young children comprising ∼5% of all pediatric cancers. MRTs are among the most genomically stable cancers, and although WTs are genomically heterogeneous, both generally lack therapeutically targetable genetic mutations. METHODS: Comparative protein activity analysis of MRTs (n = 68) and WTs (n = 132) across TCGA and TARGET cohorts, using metaVIPER, revealed elevated exportin 1 (XPO1) inferred activity. In vitro studies were performed on a panel of MRT and WT cell lines to evaluate effects on proliferation and cell-cycle progression following treatment with the selective XPO1 inhibitor selinexor. In vivo anti-tumor activity was assessed in patient-derived xenograft (PDX) models of MRTs and WTs. FINDINGS: metaVIPER analysis identified markedly aberrant activation of XPO1 in MRTs and WTs compared with other tumor types. All MRT and most WT cell lines demonstrated baseline, aberrant XPO1 activity with in vitro sensitivity to selinexor via cell-cycle arrest and induction of apoptosis. In vivo, XPO1 inhibitors significantly abrogated tumor growth in PDX models, inducing effective disease control with sustained treatment. Corroborating human relevance, we present a case report of a child with multiply relapsed WTs with prolonged disease control on selinexor. CONCLUSIONS: We report on a novel systems-biology-based comparative framework to identify non-genetically encoded vulnerabilities in genomically quiescent pediatric cancers. These results have provided preclinical rationale for investigation of XPO1 inhibitors in an upcoming investigator-initiated clinical trial of selinexor in children with MRTs and WTs and offer opportunities for exploration of inferred XPO1 activity as a potential predictive biomarker for response. FUNDING: This work was funded by CureSearch for Children's Cancer, Alan B. Slifka Foundation, NIH (U01 CA217858, S10 OD012351, and S10 OD021764), Michael's Miracle Cure, Hyundai Hope on Wheels, Cannonball Kids Cancer, Conquer Cancer the ASCO Foundation, Cycle for Survival, Paulie Strong Foundation, and the Grayson Fund.

Dual Fluorescence Isogenic Synthetic Lethal Kinase Screen and High-Content Secondary Screening for MUC16/CA125-Selective Agents.

Significant strides have been made in the development of precision therapeutics for cancer. Aberrantly expressed glycoproteins represent a potential avenue for therapeutic development. The MUC16/CA125 glycoprotein serves as a biomarker of disease and a driver of malignant transformation in epithelial ovarian cancer. Previously, we demonstrated a proof-of-principle approach to selectively targeting MUC16+ cells. In this report, we performed a synthetic lethal kinase screen using a human kinome RNAi library and identified key pathways preferentially targetable in MUC16+ cells using isogenic dual-fluorescence ovarian cancer cell lines. Using a separate approach, we performed high-content small-molecule screening of six different libraries of 356,982 compounds for MUC16/CA125-selective agents and identified lead candidates that showed preferential cytotoxicity in MUC16+ cells. Compounds with differential activity were selected and tested in various other ovarian cell lines or isogenic pairs to identify lead compounds for structure-activity relationship (SAR) selection. Lead siRNA and small-molecule inhibitor candidates preferentially inhibited invasion of MUC16+ cells in vitro and in vivo, and we show that this is due to decreased activation of MAPK, and non-receptor tyrosine kinases. Taken together, we present a comprehensive screening approach to the development of a novel class of MUC16-selective targeted therapeutics and identify candidates suitable for further clinical development.

Translational Strategies for Repotrectinib in Neuroblastoma.

Limited clinical data are available regarding the utility of multikinase inhibition in neuroblastoma. Repotrectinib (TPX-0005) is a multikinase inhibitor that targets ALK, TRK, JAK2/STAT, and Src/FAK, which have all been implicated in the pathogenesis of neuroblastoma. We evaluated the preclinical activity of repotrectinib monotherapy and in combination with chemotherapy as a potential therapeutic approach for relapsed/refractory neuroblastoma. In vitro sensitivity to repotrectinib, ensartinib, and cytotoxic chemotherapy was evaluated in neuroblastoma cell lines. In vivo antitumor effect of repotrectinib monotherapy, and in combination with chemotherapy, was evaluated using a genotypically diverse cohort of patient-derived xenograft (PDX) models of neuroblastoma. Repotrectinib had comparable cytotoxic activity across cell lines irrespective of ALK mutational status. Combination with chemotherapy demonstrated increased antiproliferative activity across several cell lines. Repotrectinib monotherapy had notable antitumor activity and prolonged event-free survival compared with vehicle and ensartinib in PDX models (P < 0.05). Repotrectinib plus chemotherapy was superior to chemotherapy alone in ALK-mutant and ALK wild-type PDX models. These results demonstrate that repotrectinib has antitumor activity in genotypically diverse neuroblastoma models, and that combination of a multikinase inhibitor with chemotherapy may be a promising treatment paradigm for translation to the clinic.

Comprehensive Molecular Profiling of Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is characterized by the EWSR1-WT1 t(11;22) (p13:q12) translocation. Few additional putative drivers have been identified, and research has suffered from a lack of model systems. Next-generation sequencing (NGS) data from 68 matched tumor-normal samples, whole-genome sequencing data from 10 samples, transcriptomic and affymetrix array data, and a bank of DSRCT patient-derived xenograft (PDX) are presented. EWSR1-WT1 fusions were noted to be simple, balanced events. Recurrent mutations were uncommon, but were noted in TERT (3%), ARID1A (6%), HRAS (5%), and TP53 (3%), and recurrent loss of heterozygosity (LOH) at 11p, 11q, and 16q was identified in 18%, 22%, and 34% of samples, respectively. Comparison of tumor-normal matched versus unmatched analysis suggests overcalling of somatic mutations in prior publications of DSRCT NGS data. Alterations in fibroblast growth factor receptor 4 (FGFR4) were identified in 5 of 68 (7%) of tumor samples, whereas differential overexpression of FGFR4 was confirmed orthogonally using 2 platforms. PDX models harbored the pathognomic EWSR1-WT1 fusion and were highly representative of corresponding tumors. Our analyses confirm DSRCT as a genomically quiet cancer defined by the balanced translocation, t(11;22)(p13:q12), characterized by a paucity of secondary mutations but a significant number of copy number alterations. Against this genomically quiet background, recurrent activating alterations of FGFR4 stood out, and suggest that this receptor tyrosine kinase, also noted to be highly expressed in DSRCT, should be further investigated. Future studies of DSRCT biology and preclinical therapeutic strategies should benefit from the PDX models characterized in this study. IMPLICATIONS: These data describe the general quiescence of the desmoplastic small round cell tumor (DSRCT) genome, present the first available bank of DSRCT model systems, and nominate FGFR4 as a key receptor tyrosine kinase in DSRCT, based on high expression, recurrent amplification, and recurrent activating mutations.

Antibodies Against Specific MUC16 Glycosylation Sites Inhibit Ovarian Cancer Growth.

Expression of the retained C-terminal extracellular portion of the ovarian cancer glycoprotein MUC16 induces transformation and tumor growth. However, the mechanisms of MUC16 oncogenesis related to glycosylation are not clearly defined. We establish that MUC16 oncogenic effects are mediated through MGAT5-dependent N-glycosylation of two specific asparagine sites within its 58 amino acid ectodomain. Oncogenic signaling from the C-terminal portion of MUC16 requires the presence of Galectin-3 and growth factor receptors colocalized on lipid rafts. These effects are blocked upon loss of either Galectin-3 expression or activity MGAT5. Using synthetic MUC16 glycopeptides, we developed novel N-glycosylation site directed monoclonal antibodies that block Galectin-3-mediated MUC16 interactions with cell surface signaling molecules. These antibodies inhibit invasion of ovarian cancer cells, directly blocking the in vivo growth of MUC16-bearing ovarian cancer xenografts, elucidating new therapeutic modalities.

Expression of the Carboxy-Terminal Portion of MUC16/CA125 Induces Transformation and Tumor Invasion.

The CA125 antigen is found in the serum of many patients with serous ovarian cancer and has been widely used as a disease marker. CA125 has been shown to be an independent factor for clinical outcome in this disease. In The Cancer Genome Atlas ovarian cancer project, MUC16 expression levels are frequently increased, and the highest levels of MUC16 expression are linked to a significantly worse survival. To examine the biologic effect of the proximal portion of MUC16/CA125, NIH/3T3 (3T3) fibroblast cell lines were stably transfected with the carboxy elements of MUC16. As few as 114 amino acids from the carboxy-terminal portion of MUC16 were sufficient to increase soft agar growth, promote matrigel invasion, and increase the rate of tumor growth in athymic nude mice. Transformation with carboxy elements of MUC16 was associated with activation of the AKT and ERK pathways. MUC16 transformation was associated with up-regulation of a number of metastases and invasion gene transcripts, including IL-1ß, MMP2, and MMP9. All observed oncogenic changes were exclusively dependent on the extracellular "ectodomain" of MUC16. The biologic impact of MUC16 was also explored through the creation of a transgenic mouse model expressing 354 amino acids of the carboxy-terminal portion of MUC16 (MUC16c354). Under a CMV, early enhancer plus chicken ß actin promoter (CAG) MUC16c354 was well expressed in many organs, including the brain, colon, heart, kidney, liver, lung, ovary, and spleen. MUC16c354 transgenic animals appear to be viable, fertile, and have a normal lifespan. However, when crossed with p53-deficient mice, the MUC16c354:p53+/- progeny displayed a higher frequency of spontaneous tumor development compared to p53+/- mice alone. We conclude that the carboxy-terminal portion of the MUC16/CA125 protein is oncogenic in NIH/3T3 cells, increases invasive tumor properties, activates the AKT and ERK pathways, and contributes to the biologic properties of ovarian cancer.

Dual-fluorescence isogenic high-content screening for MUC16/CA125 selective agents.

Most of the currently used cancer chemotherapies are based on compounds that inhibit general cellular mechanisms, such as DNA replication or tubulin function, and lack specificity in relation to features of the cancer cell. Recent advances in genomic studies have increased our knowledge of tumor cell biology, and a panoply of new targets have been postulated. This has provided an opportunity to develop and validate drugs that specifically target cancer cells through their unique genetic characteristics. Identification of MUC16/CA125 both as a marker and a driver of transformation led us to design a target-based high-content screen to identify and classify compounds that exhibit differential effect on MUC16-expressing cells. We developed a coculture assay in 384-well plate containing isogenic ovarian cancer cells that are positive or negative for the MUC16 protein. High-throughput screening of our small molecule pilot library led to the identification of compounds preferentially cytotoxic to MUC16(+) or MUC16(-) cells, using a Preferential Score analysis. We compared screening results in both A2780 and SK-OV-3 ovarian cancer cells in single and coculture settings. We also identified compounds that were cytotoxic for both types of ovarian cancer cells regardless of the MUC16 status. Compounds that were preferentially targeting MUC16 cells were subsequently confirmed by caspase-induction assays. The isogenic, dual-color fluorescence strategy is an innovative approach that can effectively identify novel drug candidates, selectively targeting cancer cells that have unique molecular properties.

Carotenogénesis de cinco cepas del alga Dunaliella sp. (Chlorophyceae) aisladas de lagunas hipersalinas de Venezuela / Carotenogenesis of five strains of the algae Dunaliella sp. (Chlorophyceae) isolated from Venezuelan hypersaline lagoons

Se realizaron cultivos discontinuos (medio Algal con 0.5 mM de NaNO3 y 27% de NaCl) de cinco cepas de Dunaliella sp., aisladas de diferentes lagunas hipersalinas de Venezuela (Araya, Coche, Peonía, Cumaraguas y Boca Chica) y una cepa de referencia (Dunaliella salina LB1644). Los bioensayos se mantuvieron a 25 ± 1 °C con aireación constante, fotoperiodo 12:12 y dos intensidades luminosas (195 y 390 µE.m-2.s-1) durante 30 días. El crecimiento celular se determinó diariamente mediante conteo celular en cámara de Neubaüer. La clorofila a y los carotenoides totales se analizaron al final del ensayo. Las mayores densidades celulares correspondieron a los ensayos de menor intensidad luminosa. La cepa que alcanzó la mayor densidad celular fue la aislada de Boca Chica (8 x106 y 2.5 x106 cel.ml-1 a 195 y 390 µE.m-2.s-1, respectivamente). El incremento de la intensidad luminosa en los cultivos produjo una disminución significativa de las tasas de crecimiento en todas las cepas. Los carotenoides totales por volumen fueron mayores a 390 µE.m-2 .s-1; siendo las cepas de referencia LB1644, Coche y Araya las que produjeron mayor cantidad (38.4; 32.8 y 21.0 µg.ml-1, respectivamente). El contenido de carotenoides totales por célula en los dos tratamientos fue significativamente diferente, obteniéndose la mayor concentración a 390 µE.m-2.s-1. Las cepas LB1644 y Coche fueron las que produjeron los valores más altos de carotenos (137.14 y 106.06 pg.cel-1, respectivamente). La cepa LB1644 presentó la mayor relación carotenoides totales:clorofila a (20:1) a 195 µE.m-2.s-1, mientras que en la cepa Coche no se evidenciaron diferencias significativas entre las dos intensidades (15:1). El resto de las cepas mostraron relaciones inferiores a uno. Nuestros resultados sugieren que las cepas Coche y Araya pueden ser potencialmente utilizadas en la biotecnología de producción de carotenoides

We evaluated discontinuous cultures (Algal medium at 0.5 mM of NaNO3 , and 27% NaCl) of five strains of Dunaliella sp. isolated from Venezuelan hypersaline lagoons (Araya, Coche, Peonía, Cumaraguas, and Boca Chica) and one strain from a reference collection (Dunaliella salina, LB1644). Cultures were maintained to 25±1 °C, with constant aeration, photoperiod 12:12, and two light intensities (195 and 390 µE.m-2 .s-1) during 30 days. Cell count was recorded on a daily basis using a Neubaüer camera. Totals of chlorophyll a and carotenoids were measured at the end of the experiment. The largest cellular densities were measured during the smallest light intensities. The strain with the largest cellular density was isolated from Boca Chica (8 x106 and 2.5 x106 cel.ml-1 a 390 and 195µE.m-2 .s-1, respectively). The increment of light intensity produced a significant reduction of growth rates in all strains. Totals of carotenoids by volume were as large as 390 µE.m-2 .s-1. Strains LB1644, from Coche and Araya were those that produced the largest amount of carotenoids (38.4; 32.8 and 21.0 µg.ml-1 , respectively). Differences total carotenoids by cell between treatments were significant. The largest concentration was 390 µE.m-2 .s-1 . The strains LB1644 and Coche produced the highest values of carotenes (137.14 and 106.06 pg.cel-1, respectively). Differences in the relation carotenoid:chlorophyll a between the strains at various light intensities was significant. Strains LB1644 presented the largest value of the relation carotenoids:chlorophyll a (20:1) at 195 µE.m-2 .s-1. No significant differences were detected in the strain Coche (15:1). All the other strains showed relations lower than one. Our results suggest that the strains of Coche and Araya show potential to be used in the biotechnology of carotenoids production

[Carotenogenesis of five strains of the algae Dunaliella sp. (Chlorophyceae) isolated from Venezuelan hypersaline lagoons]. / Carotenogénesis de cinco cepas del alga Dunaliella sp. (chlorophyceae) aisladas de lagunas hipersalinas de Venezuela.

We evaluated discontinuous cultures (Algal medium at 0.5 mM of NaNO3, and 27% NaCI) of five strains of Dunaliella sp. isolated from Venezuelan hypersaline lagoons (Araya, Coche, Peonia, Cumaraguas. and Boca Chica) and one strain from a reference collection (Dunaliella salina, LB1644). Cultures were maintained to 25+/-1 degrees C, with constant aeration, photoperiod 12:12, and two light intensities (195 and 390 microE.m(-2).s(-1)) during 30 days. Cell count was recorded on a daily basis using a Neubaüer camera. Totals of chlorophyll a and carotenoids were measured at the end of the experiment. The largest cellular densities were measured during the smallest light intensities. The strain with the largest cellular density was isolated from Boca Chica (8 xl0(6) and 2.5 xl0(6) cel.ml(-1) a 390 and 195microE.m(-2).s(-1), respectively). The increment of light intensity produced a significant reduction of growth rates in all strains. Totals of carotenoids by volume were as large as 390 microE.m(-2).s(-1). Strains LB 1644, from Coche and Araya were those that produced the largest amount of carotenoids (38.4; 32.8 and 21.0 microg.ml(-1), respectively). Differences total carotenoids by cell between treatments were significant. The largest concentration was 390 microE.m(-2).s(-1). The strains LB 1644 and Coche produced the highest values of carotenes (137.14 and 106.06 pg.cel(-1), respectively). Differences in the relation carotenoid:chlorophyll a between the strains at various light intensities was significant. Strains LB1644 presented the largest value of the relation carotenoids:chlorophyll a (20:1) at 195 microE.m(-2).s(-1). No significant differences were detected in the strain Coche (15:1). All the other strains showed relations lower than one. Our results suggest that the strains of Coche and Araya show potential to be used in the biotechnology of carotenoids production.