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The RAS/MAPK pathway is an emerging targeted pathway across a spectrum of both adult and pediatric cancers. Typically, this is associated with a single, well-characterized point mutation in an oncogene. Hypermutant tumors that harbor many somatic mutations may obscure the interpretation of such targetable genomic events. We find that replication repair–deficient (RRD) cancers, which are universally hypermutant and affect children born with RRD cancer predisposition, are enriched for RAS/MAPK mutations (P = 10−8). These mutations are not random, exist in subclones, and increase in allelic frequency over time. The RAS/MAPK pathway is activated both transcriptionally and at the protein level in patient-derived RRD tumors, and these tumors responded to MEK inhibition in vitro and in vivo. Treatment of patients with RAS/MAPK hypermutant gliomas reveals durable responses to MEK inhibition. Our observations suggest that hypermutant tumors may be addicted to oncogenic pathways, resulting in favorable response to targeted therapies.Significance:Tumors harboring a single RAS/MAPK driver mutation are targeted individually for therapeutic purposes. We find that in RRD hypermutant cancers, mutations in the RAS/MAPK pathway are enriched, highly expressed, and result in sensitivity to MEK inhibitors. Targeting an oncogenic pathway may provide therapeutic options for these hypermutant polyclonal cancers.This article is highlighted in the In This Issue feature, p. 1307

Table S1: Summary of likely pathogenic mutations detected in Foundation Medicine cohort of 1215 pediatric cancers. Table S2: Summary of RAS/MAPK pathway mutations and consequences detected in 48 RRD cancers.

Supplementary Figure S1 (related to Figure 1): PoleP286R mouse model design, validation, and tumor findings. Supplementary Figure S2 (related to Figure 1): PoleS459F mouse model design, validation, and tumor findings. Supplementary Figure S3 (related to Figure 2): WES analysis reveals a stochastic accumulation of mutations in Pole mutant tumors. Supplementary Figure S4 (related to Figure 2): Tumors from Pole mutant mice exhibit mutational signatures found in POLE driven human cancers. Supplementary Figure S5 (related to Figure 2): Exome data from tumor fractions from a single mouse were compared for 3 additional mice. Supplementary Figure S6. Determination of cell of origin for PoleS459F/S459F and PoleS459F/+ mice lymphomas (Related to Figure 3). Supplementary Figure S7. Characterization of T cell malignancies in PoleS459F/S459F and PoleS459F/+ mice (Related to Figure 3). Supplemental Figure S8. Extrinsic effect of 'Group B' malignant T cells on B cell population; (Related to Figure 3).

POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype–phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo, independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole-driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE-driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients.Significance:Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy.See related commentary by Wisdom and Kirsch p. 5459

CyTOF panel antibodies

Metastatic relapse is the major cause of death in pediatric neuroblastoma, where there remains a lack of therapies to target this stage of disease. To understand the molecular mechanisms mediating neuroblastoma metastasis, we developed a mouse model using intracardiac injection and in vivo selection to isolate malignant cell subpopulations with a higher propensity for metastasis to bone and the central nervous system. Gene expression profiling revealed primary and metastatic cells as two distinct cell populations defined by differential expression of 412 genes and of multiple pathways, including CADM1, SPHK1, and YAP/TAZ, whose expression independently predicted survival. In the metastatic subpopulations, a gene signature was defined (MET-75) that predicted survival of neuroblastoma patients with metastatic disease. Mechanistic investigations demonstrated causal roles for CADM1, SPHK1, and YAP/TAZ in mediating metastatic phenotypes in vitro and in vivo. Notably, pharmacologic targeting of SPHK1 or YAP/TAZ was sufficient to inhibit neuroblastoma metastasis in vivo. Overall, we identify gene expression signatures and candidate therapeutics that could improve the treatment of metastatic neuroblastoma. Cancer Res; 77(3); 696–706. ©2017 AACR.

Further details of materials & methods provided in supplementary data.

Supplemental Titles and Figure Legends

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Supplementary Figure 1 - The metastatic subpopulations display enhanced metastatic burden relative to the SK-N-AS-TR parental cell line in vivo and increased chemoresistance in vitro. Supplementary Figure 2 - GSEA enrichment map show signaling pathways that are differentially regulated between parental and metastatic subtype. Supplementary Figure 3 - Genes identified in enhanced metastatic subpopulations predict survival of human NB patients in multiple datasets. Supplementary Figure 4 - CADM1 over-expression decreases migration of SHEP cell line. Supplementary Figure 5 - Verteporfin (VP), a pharmacological inhibitor of YAP, induces apoptosis and decreases migration of metastatic cells in in vitro and inhibits the metastatic phenotype in vivo. Supplementary Figure 6 - Pharmacologic inhibition of SPHK1 decreases the viability of NB cell lines and STAT3 and NFkB signaling in the enhanced metastatic subpopulations. Supplementary Figure 7 - Metastatic signature (MET-75) predicts poor survival in MYCN non-amplified patients and outperforms randomly generated 75-gene signatures.

Supplementary Table 1 - Summary of metastatic lesions in mice injected with the parental and enhanced metastatic subpopulations of SK-N-AS-TR.

Supplementary Figure 1 from Identification of a Novel c-Myc Protein Interactor, JPO2, with Transforming Activity in Medulloblastoma Cells

c-myc oncogene activation is critical in the pathogenesis of a spectrum of human malignancies. The c-Myc NH2-terminal domain (MycNTD) is essential for cellular transformation, and mediates critical protein interactions that modulate c-Myc oncogenic properties. In medulloblastoma, the most common malignant pediatric brain tumor, deregulated c-myc expression is linked with poorer disease phenotypes and outcomes. The biological basis for these associations is, however, not well understood. To better understand mechanisms underlying Myc-mediated transformation of medulloblastoma, we sought to identify novel MycNTD protein interactors from a medulloblastoma cell line library using a unique two-hybrid system. We identified a novel MycNTD binding protein, JPO2, which shows nuclear colocalization with c-Myc, and interacts with c-Myc both in vitro and in mammalian cells. In Rat1a transformation assays, JPO2 potentiates c-Myc transforming activity, and can complement a transformation-defective Myc mutant. Immunohistochemical studies indicate tumor-specific JPO2 expression in human medulloblastoma, and an association of JPO2 expression with metastatic tumors. Significantly, JPO2 expression induces colony formation in UW228, a medulloblastoma cell line, whereas RNAi-mediated JPO2 knockdown impairs colony formation in UW228, and in Myc-transformed UW228 cells. These data provide evidence for biochemical and functional interaction between c-Myc and JPO2 in medulloblastoma transformation. JPO2 is closely related to JPO1, a Myc transcriptional target with transforming activity. As tumor-specific JPO1 expression in human and murine medulloblastoma has also been reported; these collective observations suggest important functional links between the novel JPO protein family and c-Myc in medulloblastoma transformation.

The use of frozen section histological analysis in primary obstetrical brachial plexus palsy reconstruction, though widespread, is not universally practiced. Our objective was to develop a histological grading scale that could be used to determine whether further resection of a microscopically suboptimal, though grossly satisfactory stump could lead to a measurable improvement in histological appearance. A 13-point grading tool assessing attributes of the epineurium, perineurium, and endoneurium was tested for interrater reliability. The histological appearance of initial nerve biopsies and of subsequent nerve reexcisions stained with toluidine blue was reviewed retrospectively (n = 52). Specimens were graded in a blinded fashion by a neuropathologist and a medical student. There was high agreement between expert and novice global rating scores with an intraclass correlation coefficient of 0.89 (95% confidence interval 0.85 to 0.93). A comparison of scores between subsequent sections of the same nerve stump revealed a significant decrease of 3.00 (expert) and 2.00 (novice) points ( P < 0.001) in the median global rating score, demonstrating improvement in histological grade. The novel grading tool was used to demonstrate that recutting a microscopically poor, though grossly acceptable nerve stump in obstetrical palsy surgery can yield a significantly improved histological grade.

Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However, resistance inevitably emerges against these therapies, particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets.High content screening of a 129 compound library identified agents that inhibited SHH MB growth. Lead molecular target levels, p90 ribosomal S6 kinase (RSK) were characterized by immunoblotting and qRT-PCR. Comparisons were made to human neural stem cells (hNSC). Impact of inhibiting RSK with the small molecule BI-D1870 or siRNA was assessed in growth assays (monolayer, neurosphere, and soft agar). NanoString was used to detect RSK in a cohort of 66 patients with MB. To determine BI-D1870 pharmacokinetics/pharmacodynamics, 100 mg/kg was I.P. injected into mice and tissues were collected at various time points.Daoy, ONS76, UW228, and UW426 MB cells were exquisitely sensitive to BI-D1870 but unresponsive to SHH inhibitors. Anti-tumor growth corresponded with inactivation of RSK in MB cells. BI-D1870 had no effect on hNSCs. Inhibiting RSK with siRNA or BI-D1870 suppressed growth, induced apoptosis, and sensitized cells to SHH agents. Notably, RSK expression is correlated with SHH patients. In mice, BI-D1870 was well-tolerated and crossed the blood-brain barrier (BBB).RSK inhibitors are promising because they target RSK which is correlated with SHH patients as well as cause high levels of apoptosis to only MB cells. Importantly, BI-D1870 crosses the BBB, acting as a scaffold for development of more long-lived RSK inhibitors.

Cerebral hyaline astrocytic inclusions have been observed in a subset of patients with early onset epilepsy, brain structural anomalies, and developmental delay, which indicates that it may represent a unique clinicopathologic entity. To further characterize this condition we use proteomics to investigate differentially expressed proteins in epileptic brain tissue from three pediatric epileptic patients with cerebral hyaline astrocytic inclusions, ranging in age from 5-13 years, and compare to brain tissue from two normal controls. Catalase and carbonic anhydrase I both exhibited increased expression in epileptic brain tissue compared to controls. These findings were confirmed by Western blot analysis. Furthermore, both proteins were localized to astrocytes and in epileptic brain were located within the cerebral hyaline astrocytic inclusions, suggesting a potential role in the generation of this pathologic feature of early onset epilepsy with cerebral hyaline astrocytic inclusions.

The authors report on 2 newborn infants with the unusual presentation of intrinsic brainstem tumors. Both nondysmorphic, full-term neonates had cranial nerve palsies and hypotonia. Diagnoses of diffuse intrinsic brainstem gliomas were made on the basis of magnetic resonance imaging, which showed large expansive, nonenhancing intrinsic pontine masses. Intrinsic pontine tumors, characteristically seen in school-age children, are most often high-grade gliomas that are almost invariably fatal. However, the microanatomy and natural history of pontine tumors in neonates are unknown. With parental consent, both newborns were treated expectantly with supportive care but died of progressive disease by 2 weeks of age. In one child, postmortem examination revealed a primary brainstem primitive neuroectodermal tumor. The authors conclude that, as in older children, neonatal intrinsic brainstem tumors may be of a highly malignant nature. The rapid tumor progression in both cases indicates that where a diagnostic procedure may pose significant risks, supportive observation can aid in distinguishing malignant from benign tumor growth.

Chlorine oxidants (chlorine gas, sodium hypochlorite, and calcium hypochlorite) are used as biocides to control fouling in seawater cooled power generating plants and to kill pathogens in sewage effluents entering estuarine waters. Organisms killed include algae, fungi, bacteria, barnacles, oysters, and clams. Use of these chemicals in saline waters results in the formation of halogenated by-products (JOHANNESSON 1958, DUURSAMPARSI 1976, DOVE 1973). BEAN et al, (1978), in a study of chlorinated seawater, identified eighteen organic compounds generated by chlorination. Due to rapid substitution of bromine for chlorine, brominated rather than chlorinated by-products are formed (FARKAS et al. 1949, GALAL-GORCHEVMORRIS 1965, MACALADY et al. )977, SUGAMHELZ 1977). The effects of these chlorinated by-products on estuarine phytoplankton are not known. Experiments in this study examined singly the effects of fifteen commercially available compounds on photosynthesis by estuarine phytoplankton.

Three species of marine algae representing major taxonomic groups of phytoplankton, Isochrysis galbana (Chrysophyceae), Carteria sp. (Chlorophyceae), and Thalassiosira pseudonana (Bacillariophyceae), were utilized to investigate the potential of naturally occurring chlorophyll a of living algae to produce trihalomethanes during the chlorination of saline waters. Chlorination of filtered natural estuarine water (salinity=23 p.p.t.) from the North Edisto River, South Carolina, results in rapid formation of 201–221 μg 1 −1 trihalomethanes comprised mainly of bromoform (CHBr 3 ) and chlorodibromomethane (CHBr 2 Cl). In the presence of 10 6 cells ml −1 Isochrysis galbana , chlorination of filtered estuarine water with sodium hypochlorite (NaOCl) to a nominal 10 mg 1 −1 chlorine increased the total trihalomethane concentration by an average of 41% ( N = 6). The presence of Thalassiosira pseudonana resulted in an average 24% decrease ( N = 6) while Carteria sp. did not produce a statistically significant effect upon the total trihalomethane concentration formed. The absence of any significant statistical correlations between the chlorophyll a content of algal cultures and trihalomethane concentrations causes us to discount chlorine-chlorophyll a interactions as a source of these compounds. However, trihalomethane concentrations produced from the chlorination of algal culture media, after removal of algal populations, suggests instead that the observed trends in trihalomethane production are mainly due to chlorines reaction with the by-products of algal metabolism.