CD57 defines a novel cancer stem cell that drive invasion of diffuse pediatric-type high grade gliomas.

BACKGROUND: Diffuse invasion remains a primary cause of treatment failure in pediatric high-grade glioma (pHGG). Identifying cellular driver(s) of pHGG invasion is needed for anti-invasion therapies. METHODS: Ten highly invasive patient-derived orthotopic xenograft (PDOX) models of pHGG were subjected to isolation of matching pairs of invasive (HGGINV) and tumor core (HGGTC) cells. RESULTS: pHGGINV cells were intrinsically more invasive than their matching pHGGTC cells. CSC profiling revealed co-positivity of CD133 and CD57 and identified CD57+CD133- cells as the most abundant CSCs in the invasive front. In addition to discovering a new order of self-renewal capacities, i.e., CD57+CD133- > CD57+CD133+ > CD57-CD133+ > CD57-CD133- cells, we showed that CSC hierarchy was impacted by their spatial locations, and the highest self-renewal capacities were found in CD57+CD133- cells in the HGGINV front (HGGINV/CD57+CD133- cells) mediated by NANOG and SHH over-expression. Direct implantation of CD57+ (CD57+/CD133- and CD57+/CD133+) cells into mouse brains reconstituted diffusely invasion, while depleting CD57+ cells (i.e., CD57-CD133+) abrogated pHGG invasion. CONCLUSION: We revealed significantly increased invasive capacities in HGGINV cells, confirmed CD57 as a novel glioma stem cell marker, identified CD57+CD133- and CD57+CD133+ cells as a new cellular driver of pHGG invasion and suggested a new dual-mode hierarchy of HGG stem cells.

Targeting GBM with an Oncolytic Picornavirus SVV-001 alone and in combination with fractionated Radiation in a Novel Panel of Orthotopic PDX models.

BACKGROUND: Animal models representing different molecular subtypes of glioblastoma multiforme (GBM) is desired for developing new therapies. SVV-001 is an oncolytic virus selectively targeting cancer cells. It's capacity of passing through the blood brain barrier makes is an attractive novel approach for GBM. MATERIALS AND METHODS: 23 patient tumor samples were implanted into the brains of NOD/SCID mice (1 × 105 cells/mouse). Tumor histology, gene expression (RNAseq), and growth rate of the developed patient-derived orthotopic xenograft (PDOX) models were compared with the originating patient tumors during serial subtransplantations. Anti-tumor activities of SVV-001 were examined in vivo; and therapeutic efficacy validated in vivo via single i.v. injection (1 × 1011 viral particle) with or without fractionated (2 Gy/day x 5 days) radiation followed by analysis of animal survival times, viral infection, and DNA damage. RESULTS: PDOX formation was confirmed in 17/23 (73.9%) GBMs while maintaining key histopathological features and diffuse invasion of the patient tumors. Using differentially expressed genes, we subclassified PDOX models into proneural, classic and mesenchymal groups. Animal survival times were inversely correlated with the implanted tumor cells. SVV-001 was active in vitro by killing primary monolayer culture (4/13 models), 3D neurospheres (7/13 models) and glioma stem cells. In 2/2 models, SVV-001 infected PDOX cells in vivo without harming normal brain cells and significantly prolonged survival times in 2/2 models. When combined with radiation, SVV-001 enhanced DNA damages and further prolonged animal survival times. CONCLUSION: A panel of 17 clinically relevant and molecularly annotated PDOX modes of GBM is developed, and SVV-001 exhibited strong anti-tumor activities in vitro and in vivo.

Evaluation of an EZH2 inhibitor in patient-derived orthotopic xenograft models of pediatric brain tumors alone and in combination with chemo- and radiation therapies.

Brain tumors are the leading cause of cancer-related death in children. Tazemetostat is an FDA-approved enhancer of zeste homolog (EZH2) inhibitor. To determine its role in difficult-to-treat pediatric brain tumors, we examined EZH2 levels in a panel of 22 PDOX models and confirmed EZH2 mRNA over-expression in 9 GBM (34.6 ± 12.7-fold) and 11 medulloblastoma models (6.2 ± 1.7 in group 3, 6.0 ± 2.4 in group 4) accompanied by elevated H3K27me3 expression. Therapeutic efficacy was evaluated in 4 models (1 GBM, 2 medulloblastomas and 1 ATRT) via systematically administered tazemetostat (250 and 400 mg/kg, gavaged, twice daily) alone and in combination with cisplatin (5 mg/kg, i.p., twice) and/or radiation (2 Gy/day × 5 days). Compared with the untreated controls, tazemetostat significantly (Pcorrected < 0.05) prolonged survival times in IC-L1115ATRT (101% at 400 mg/kg) and IC-2305GBM (32% at 250 mg/kg, 45% at 400 mg/kg) in a dose-dependent manner. The addition of tazemetostat with radiation was evaluated in 3 models, with only one [IC-1078MB (group 4)] showing a substantial, though not statistically significant, prolongation in survival compared to radiation treatment alone. Combining tazemetostat (250 mg/kg) with cisplatin was not superior to cisplatin alone in any model. Analysis of in vivo drug resistance detected predominance of EZH2-negative cells in the remnant PDOX tumors accompanied by decreased H3K27me2 and H3K27me3 expressions. These data supported the use of tazemetostat in a subset of pediatric brain tumors and suggests that EZH2-negative tumor cells may have caused therapy resistance and should be prioritized for the search of new therapeutic targets.

Preservation of KIT genotype in a novel pair of patient-derived orthotopic xenograft mouse models of metastatic pediatric CNS germinoma.

Metastatic intracranial germinoma is difficult to treat. Although the proto-oncogene KIT is recognized as one of the most frequent genetic abnormalities in CNS germinoma, the development of new target therapeutic agents for CNS germinoma is hampered by the lack of clinically-relevant animal models that replicate the mutated or over-expressed KIT. CNS germinoma tumor cells from five pediatric patients were directly implanted into the brains of Rag2/severe combined immune deficiency mice. Once established, the xenograft tumors were sub-transplanted in vivo in mouse brains. Characterization of xenograft tumors were performed through histologic and immunohistochemical staining, and KIT mutation analysed with quantitative pyro-sequencing. Expression of putative cancer stem cell markers (CD133, CD15, CD24, CD44, CD49f) was analyzed through flow cytometry. Two patient-derived orthotopic xenograft (PDOX) models (IC-6999GCT and IC-9302GCT) were established from metastatic germinoma and serially sub-transplanted five times in mouse brains. Similar to the original patient tumors, they both exhibited faint expression (+) of PLAP, no expression (-) of ß-HCG and strong (+++) expression of KIT. KIT mutation (D816H), however, was only found in IC-9320GCT. This mutation was maintained during the five in vivo tumor passages with an increased mutant allele frequency compared to the patient tumor. Expression of putative cancer stem cell markers CD49f and CD15 was also detected in a small population of tumor cells in both models. This new pair of PDOX models replicated the key biological features of pediatric intracranial germinoma and should facilitate the biological and pre-clinical studies for metastatic intracranial germinomas.

Dissection, Histological Processing, and Gene Expression Analysis of Murine Supraclavicular Brown Adipose Tissue.

Brown adipose tissue (BAT)-mediated thermogenesis plays an important role in the regulation of metabolism, and its morphology and function can be greatly impacted by environmental stimuli in mice and humans. Currently, murine interscapular BAT (iBAT), which is located between two scapulae in the upper dorsal flank of mice, is the main BAT depot used by research laboratories to study BAT function. Recently, a few previously unknown BAT depots were identified in mice, including one analogous to human supraclavicular brown adipose tissue. Unlike iBAT, murine supraclavicular brown adipose tissue (scBAT) is situated in the intermediate layer of the neck and thus cannot be accessed as readily. To facilitate the study of newly identified mouse scBAT, presented herein is a protocol detailing the steps to dissect intact scBAT from postnatal and adult mice. Due to scBAT's small size relative to other adipose depots, procedures have been modified and optimized specifically for processing scBAT. Among these modifications is the use of a dissecting microscope during tissue collection to increase the precision and homogenization of frozen scBAT samples to raise the efficiency of subsequent qPCR analysis. With these optimizations, the identification of, morphological appearance of, and molecular characterization of the scBAT can be determined in mice.

Direct Implantation of Patient Brain Tumor Cells into Matching Locations in Mouse Brains for Patient-Derived Orthotopic Xenograft Model Development.

Background: Despite multimodality therapies, the prognosis of patients with malignant brain tumors remains extremely poor. One of the major obstacles that hinders development of effective therapies is the limited availability of clinically relevant and biologically accurate (CRBA) mouse models. Methods: We have developed a freehand surgical technique that allows for rapid and safe injection of fresh human brain tumor specimens directly into the matching locations (cerebrum, cerebellum, or brainstem) in the brains of SCID mice. Results: Using this technique, we successfully developed 188 PDOX models from 408 brain tumor patient samples (both high-and low-grade) with a success rate of 72.3% in high-grade glioma, 64.2% in medulloblastoma, 50% in ATRT, 33.8% in ependymoma, and 11.6% in low-grade gliomas. Detailed characterization confirmed their replication of the histopathological and genetic abnormalities of the original patient tumors. Conclusions: The protocol is easy to follow, without a sterotactic frame, in order to generate large cohorts of tumor-bearing mice to meet the needs of biological studies and preclinical drug testing.

Cholera toxin induces a shift from inactive to active cyclooxygenase 2 in alveolar macrophages activated by Mycobacterium bovis BCG.

Intranasal vaccination stimulates formation of cyclooxygenases (COX) and release of prostaglandin E(2) (PGE(2)) by lung cells, including alveolar macrophages. PGE(2) plays complex pro- or anti-inflammatory roles in facilitating mucosal immune responses, but the relative contributions of COX-1 and COX-2 remain unclear. Previously, we found that Mycobacterium bovis BCG, a human tuberculosis vaccine, stimulated increased release of PGE(2) by macrophages activated in vitro; in contrast, intranasal BCG activated no PGE(2) release in the lungs, because COX-1 and COX-2 in alveolar macrophages were subcellularly dissociated from the nuclear envelope (NE) and catalytically inactive. This study tested the hypothesis that intranasal administration of BCG with cholera toxin (CT), a mucosal vaccine component, would shift the inactive, NE-dissociated COX-1/COX-2 to active, NE-associated forms. The results showed increased PGE(2) release in the lungs and NE-associated COX-2 in the majority of COX-2(+) macrophages. These COX-2(+) macrophages were the primary source of PGE(2) release in the lungs, since there was only slight enhancement of NE-associated COX-1 and there was no change in COX-1/COX-2 levels in alveolar epithelial cells following treatment with CT and/or BCG. To further understand the effect of CT, we investigated the timing of BCG versus CT administration for in vivo and in vitro macrophage activations. When CT followed BCG treatment, macrophages in vitro had elevated COX-2-mediated PGE(2) release, but macrophages in vivo exhibited less activation of NE-associated COX-2. Our results indicate that inclusion of CT in the intranasal BCG vaccination enhances COX-2-mediated PGE(2) release by alveolar macrophages and further suggest that the effect of CT in vivo is mediated by other lung cells.

Effect of CCL2 antisense oligodeoxynucleotides on bacterial translocation and subsequent sepsis in severely burned mice orally infected with Enterococcus faecalis.

Severely burned mice are susceptible to sepsis stemming from Enterococcus faecalis translocation due to the impaired generation of M1 macrophages (M1MΦs) in local translocation sites. In our previous studies, CCL2 has been characterized as a major effector molecule on the burn-associated generation of M2MΦs, an inhibitor cell type for resident MΦ conversion into M1MΦs. In this study, we tried to protect burned mice orally infected with E. faecalis utilizing CCL2 antisense oligodeoxynucleotides (ODNs). We show that M2MΦs in mesenteric lymph nodes (MLNs) were not demonstrated in burned mice treated with CCL2 antisense ODNs. M1MΦs were not induced by heat-killed E. faecalis from resident MΦs transwell-cultured with mesenteric lymph node macrophages (MLN-MΦs) from burned mice, while M1MΦs were induced by the same antigen from resident MΦs transwell-cultured with MΦs which were isolated from burned mice treated with CCL2 antisense ODNs. Bacterial growth in MLNs was shown in burned mice orally infected with a lethal dose of E. faecalis. However, after the same infection, sepsis did not develop in burned mice treated with CCL2 antisense ODNs. These results indicate that bacterial translocation and subsequent sepsis are controlled in burned mice orally infected with a lethal dose of E. faecalis by gene therapy utilizing CCL2 antisense ODNs.

Lack of Th17 cell generation in patients with severe burn injuries.

Immunodeficient patients with severe burn injuries are extremely susceptible to infection with Candida albicans. In addition to Th1 cells, IL-17-producing CD4(+) T cells (Th17 cells) have recently been described as an important effector cell in host anti-Candida resistance. In this study, therefore, we tried to induce Th17 cells in cultures of severely burned patient PBMC by stimulation with the C. albicans Ag (CAg). In the results, the biomarkers for Th17 cells (IL-17 production and intracellular expression of IL-17 and retinoic acid receptor-related orphan receptor γt) were not displayed by burn patient PBMC stimulated with CAg, whereas these biomarkers of Th17 cells were detected in cultures of healthy donor PBMC stimulated with CAg. Burn patient sera were shown to be inhibitory on CAg-stimulated Th17 cell generation in healthy donor PBMC cultures; however, Th17 cells were induced by CAg in healthy donor PBMC cultures supplemented with burn patient sera that were previously treated with anti-IL-10 mAb. Also, the biomarkers of Th17 cells were not induced by CAg in healthy donor PBMC cultures supplemented with rIL-10. IL-10 was detected in serum specimens derived from severely burned patients. These results indicate that Th17 cells are not generated in burn patient PBMC cultures supplemented with CAg. IL-10, produced in response to burn injuries, is shown to be inhibitory on Th17 cell generation. The high susceptibility of severely burned patients to C. albicans infection might be influenced if burn-associated IL-10 production is intervened.

Impaired ability of burn patient neutrophils to stimulate ß-defensin production by keratinocytes.

Immunosuppressive neutrophils (PMN-II) appearing in association with burn injury have a role on the increased susceptibility of burn patients to various infections. In the present study, the role of PMN-II on the production of human ß-defensins (HBDs), important molecules on host antimicrobial innate immunities, by human keratinocytes was studied. Normal human epidermal keratinocytes (NHEKs) were cultured with neutrophils (PMNs) isolated from burn patients or healthy volunteers in dual-chamber transwells. Culture fluids harvested 24 h after cultivation were assayed for HBDs using enzyme-linked immunosorbent assay. Also, these culture fluids were assayed for their antimicrobial activities by a standard colony-counting method using Pseudomonas aeruginosa. In the results, PMNs isolated from peripheral blood of burn patients were confirmed as PMN-II, because these cells produced CC-chemokine ligand 2 (CCL2), but not interleukin (IL)-12 and CC-chemokine ligand 3 (CCL3). Culture fluids of NHEK transwell-cultured with healthy PMNs exhibited strong killing activities against P. aeruginosa (96% inhibition), however, the growth of bacteria was not dramatically inhibited by the culture fluids of NHEK transwell-cultured with burn-patient PMNs (36% inhibition). IL-12 and CCL3 containing culture fluids of healthy PMNs stimulated with the bacterial antigen or rCCL3 and rIL-12 enhanced the production of HBD2 and HBD3 by NHEK, whereas CCL2 containing culture fluids of burn-patient PMN stimulated with the antigen or rCCL2 inhibited the HBD production by NHEK. These results indicate that PMN-II appearing in association with burn injury contribute to the decreased production of HBDs in thermally injured patients.

Regulation of TORC1 by MAPK Signaling Determines Sensitivity and Acquired Resistance to Trametinib in Pediatric BRAFV600E Brain Tumor Models.

PURPOSE: We investigated why three patient-derived xenograft (PDX) childhood BRAFV600E-mutant brain tumor models are highly sensitive to trametinib. Mechanisms of acquired resistance selected in situ, and approaches to prevent resistance were also examined, which may translate to both low-grade glioma (LGG) molecular subtypes. EXPERIMENTAL DESIGN: Sensitivity to trametinib [MEK inhibitor (MEKi)] alone or in combination with rapamycin (TORC1 inhibitor), was evaluated in pediatric PDX models. The effect of combined treatment of trametinib with rapamycin on development of trametinib resistance in vivo was examined. PDX tissue and tumor cells from trametinib-resistant xenografts were characterized. RESULTS: In pediatric models TORC1 is activated through ERK-mediated inactivation of the tuberous sclerosis complex (TSC): consequently inhibition of MEK also suppressed TORC1 signaling. Trametinib-induced tumor regression correlated with dual inhibition of MAPK/TORC1 signaling, and decoupling TORC1 regulation from BRAF/MAPK control conferred trametinib resistance. In mice, acquired resistance to trametinib developed within three cycles of therapy in all three PDX models. Resistance to trametinib developed in situ is tumor-cell-intrinsic and the mechanism was tumor line specific. Rapamycin retarded or blocked development of resistance. CONCLUSIONS: In these three pediatric BRAF-mutant brain tumors, TORC1 signaling is controlled by the MAPK cascade. Trametinib suppressed both MAPK/TORC1 pathways leading to tumor regression. While low-dose intermittent rapamycin to enhance inhibition of TORC1 only modestly enhanced the antitumor activity of trametinib, it prevented or retarded development of trametinib resistance, suggesting future therapeutic approaches using rapamycin analogs in combination with MEKis that may be therapeutically beneficial in both KIAA1549::BRAF- and BRAFV600E-driven gliomas.

Synergistic anti-tumor efficacy of mutant isocitrate dehydrogenase 1 inhibitor SYC-435 with standard therapy in patient-derived xenograft mouse models of glioma.

Clinical outcomes in patients with WHO grade II/III astrocytoma, oligodendroglioma or secondary glioblastoma remain poor. Isocitrate dehydrogenase 1 (IDH1) is mutated in > 70% of these tumors, making it an attractive therapeutic target. To determine the efficacy of our newly developed mutant IDH1 inhibitor, SYC-435 (1-hydroxypyridin-2-one), we treated orthotopic glioma xenograft model (IC-BT142AOA) carrying R132H mutation and our newly established orthotopic patient-derived xenograft (PDX) model of recurrent anaplastic oligoastrocytoma (IC-V0914AOA) bearing R132C mutation. In addition to suppressing IDH1 mutant cell proliferation in vitro, SYC-435 (15 mg/kg, daily x 28 days) synergistically prolonged animal survival times with standard therapies (Temozolomide + fractionated radiation) mediated by reduction of H3K4/H3K9 methylation and expression of mitochondrial DNA (mtDNA)-encoded molecules. Furthermore, RNA-seq of the remnant tumors identified genes (MYO1F, CTC1 and BCL9) and pathways (base excision repair, TCA cycle II, sirtuin signaling, protein kinase A, eukaryotic initiation factor 2 and α-adrenergic signaling) as mediators of therapy resistance. Our data demonstrated the efficacy SYC-435 in targeting IDH1 mutant gliomas when combined with standard therapy and identified a novel set of genes that should be prioritized for future studies to overcome SYC-435 resistance.

Epigenetic Alterations of Repeated Relapses in Patient-matched Childhood Ependymomas.

Recurrence is frequent in pediatric ependymoma (EPN). Our longitudinal integrated analysis of 30 patient-matched repeated relapses (3.67 ± 1.76 times) over 13 years (5.8 ± 3.8) reveals stable molecular subtypes (RELA and PFA) and convergent DNA methylation reprogramming during serial relapses accompanied by increased orthotopic patient derived xenograft (PDX) (13/27) formation in the late recurrences. A set of differentially methylated CpGs (DMCs) and DNA methylation regions (DMRs) are found to persist in primary and relapse tumors (potential driver DMCs) and are acquired exclusively in the relapses (potential booster DMCs). Integrating with RNAseq reveals differentially expressed genes regulated by potential driver DMRs (CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and potential booster DMRs (PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). DMCs predicators of relapse are also identified in the primary tumors. This study provides a high-resolution epigenetic roadmap of serial EPN relapses and 13 orthotopic PDX models to facilitate biological and preclinical studies.

Spatial Dissection of Invasive Front from Tumor Mass Enables Discovery of Novel microRNA Drivers of Glioblastoma Invasion.

Diffuse invasion is the primary cause of treatment failure of glioblastoma (GBM). Previous studies on GBM invasion have long been forced to use the resected tumor mass cells. Here, a strategy to reliably isolate matching pairs of invasive (GBMINV ) and tumor core (GBMTC ) cells from the brains of 6 highly invasive patient-derived orthotopic models is described. Direct comparison of these GBMINV and GBMTC cells reveals a significantly elevated invasion capacity in GBMINV cells, detects 23/768 miRNAs over-expressed in the GBMINV cells (miRNAINV ) and 22/768 in the GBMTC cells (miRNATC ), respectively. Silencing the top 3 miRNAsINV (miR-126, miR-369-5p, miR-487b) successfully blocks invasion of GBMINV cells in vitro and in mouse brains. Integrated analysis with mRNA expression identifies miRNAINV target genes and discovers KCNA1 as the sole common computational target gene of which 3 inhibitors significantly suppress invasion in vitro. Furthermore, in vivo treatment with 4-aminopyridine (4-AP) effectively eliminates GBM invasion and significantly prolongs animal survival times (P = 0.035). The results highlight the power of spatial dissection of functionally accurate GBMINV and GBMTC cells in identifying novel drivers of GBM invasion and provide strong rationale to support the use of biologically accurate starting materials in understanding cancer invasion and metastasis.

Impact of SCID mouse gender on tumorigenicity, xenograft growth and drug-response in a large panel of orthotopic PDX models of pediatric brain tumors.

Brain tumor is the leading cause of cancer related death in children. Clinically relevant animals are critical for new therapy development. To address the potential impact of animal gender on tumorigenicity rate, xenograft growth and in vivo drug responses, we retrospectively analyzed 99 of our established patient derived orthotopic xenograft mouse models (orthotopic PDX or PDOX). From 27 patient tumors, including 5 glioblastomas (GBMs), 11 medulloblastomas (MBs), 4 ependymomas (EPNs), 4 atypical teratoid/rhabdoid tumors (ATRTs) and 3 diffuse intrinsic pontine gliomas (DIPGs), that were directly implanted into matching locations in the brains of approximately equal numbers of male and female animals (n = 310) in age-matched (within 2-week age-difference) SCID mice, the tumor formation rate was 50.6 ± 21.5% in male and 52.7 ± 23.5% in female mice with animal survival times of 192.6 ± 31.7 days in male and 173.9 ± 34.5 days in female mice (P = 0.46) regardless of pathological diagnosis. Once established, PDOX tumors were serially subtransplanted for up to VII passage. Analysis of 1,595 mice from 59 PDOX models (18 GBMs, 18 MBs, 5 ATRTs, 6 EPNs, 7 DIPGs and 5 PENTs) during passage II and VII revealed similar tumor take rates of the 6 different tumor types between male (85.4 ± 15.5%) and female mice (84.7 ± 15.2%) (P = 0.74), and animal survival times were 96.7 ± 23.3 days in male mice and 99.7 ± 20 days in female (P = 0.25). A total of 284 mice from 7 GBM, 2 MB, 1 ATRT, 1 EPN, 2 DIPG and 1 PNET were treated with a series of standard and investigational drugs/compounds. The overall survival times were 106.9 ± 25.7 days in male mice, and 110.9 ± 31.8 days in female mice (P = 0.41), similar results were observed when different types/models were analyzed separately. In conclusion, our data demonstrated that the gender of SCID mice did not have a major impact on animal model development nor drug responses in vivo, and SCID mice of both genders are appropriate for use.

Patient-Derived Orthotopic Xenograft (PDOX) Mouse Models of Primary and Recurrent Meningioma.

BACKGROUND: Meningiomas constitute one-third of all primary brain tumors. Although typically benign, about 20% of these tumors recur despite surgery and radiation, and may ultimately prove fatal. There are currently no effective chemotherapies for meningioma. We, therefore, set out to develop patient-derived orthotopic xenograft (PDOX) mouse models of human meningioma using tumor. METHOD: Of nine patients, four had World Health Organization (WHO) grade I tumors, five had WHO grade II tumors, and in this second group two patients also had recurrent (WHO grade III) meningioma. We also classified the tumors according to our recently developed molecular classification system (Types A, B, and C, with C being the most aggressive). We transplanted all 11 surgical samples into the skull base of immunodeficient (SCID) mice. Only the primary and recurrent tumor cells from one patient-both molecular Type C, despite being WHO grades II and III, respectively-led to the formation of meningioma in the resulting mouse models. We characterized the xenografts by histopathology and RNA-seq and compared them with the original tumors. We performed an in vitro drug screen using 60 anti-cancer drugs followed by in vivo validation. RESULTS: The PDOX models established from the primary and recurrent tumors from patient K29 (K29P-PDOX and K29R-PDOX, respectively) replicated the histopathology and key gene expression profiles of the original samples. Although these xenografts could not be subtransplanted, the cryopreserved primary tumor cells were able to reliably generate PDOX tumors. Drug screening in K29P and K29R tumor cell lines revealed eight compounds that were active on both tumors, including three histone deacetylase (HDAC) inhibitors. We tested the HDAC inhibitor Panobinostat in K29R-PDOX mice, and it significantly prolonged mouse survival (p < 0.05) by inducing histone H3 acetylation and apoptosis. CONCLUSION: Meningiomas are not very amenable to PDOX modeling, for reasons that remain unclear. Yet at least some of the most malignant tumors can be modeled, and cryopreserved primary tumor cells can create large panels of tumors that can be used for preclinical drug testing.

Functional Precision Medicine Identifies New Therapeutic Candidates for Medulloblastoma.

Medulloblastoma is among the most common malignant brain tumors in children. Recent studies have identified at least four subgroups of the disease that differ in terms of molecular characteristics and patient outcomes. Despite this heterogeneity, most patients with medulloblastoma receive similar therapies, including surgery, radiation, and intensive chemotherapy. Although these treatments prolong survival, many patients still die from the disease and survivors suffer severe long-term side effects from therapy. We hypothesize that each patient with medulloblastoma is sensitive to different therapies and that tailoring therapy based on the molecular and cellular characteristics of patients' tumors will improve outcomes. To test this, we assembled a panel of orthotopic patient-derived xenografts (PDX) and subjected them to DNA sequencing, gene expression profiling, and high-throughput drug screening. Analysis of DNA sequencing revealed that most medulloblastomas do not have actionable mutations that point to effective therapies. In contrast, gene expression and drug response data provided valuable information about potential therapies for every tumor. For example, drug screening demonstrated that actinomycin D, which is used for treatment of sarcoma but rarely for medulloblastoma, was active against PDXs representing Group 3 medulloblastoma, the most aggressive form of the disease. Functional analysis of tumor cells was successfully used in a clinical setting to identify more treatment options than sequencing alone. These studies suggest that it should be possible to move away from a one-size-fits-all approach and begin to treat each patient with therapies that are effective against their specific tumor. SIGNIFICANCE: These findings show that high-throughput drug screening identifies therapies for medulloblastoma that cannot be predicted by genomic or transcriptomic analysis.

Tumor necrosis factor overcomes immune evasion in p53-mutant medulloblastoma.

Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-ß receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.

Persistent inactivation of macrophage cyclooxygenase-2 in mycobacterial pulmonary inflammation.

The induction of cyclooxygenase-2 (COX-2) in tissue macrophages (MØ) increases prostaglandin E(2) (PGE(2)) release, potentially down-regulating granulomatous inflammation. In response to Mycobacteria, local MØ express COX-2, which is either nuclear envelope (NE)-associated or NE-dissociated. Persistent mycobacterial pulmonary inflammation is characterized by alveolar MØ expressing NE-dissociated (inactive) COX-2 without release of PGE(2). In this study, we examined COX-2 in alveolar MØ after intranasal exposure to heat-killed Mycobacterium bovis BCG (HK-BCG). After administration, whole lungs of C57Bl/6 mice were lavaged with saline; COX-2 expression and PGE(2) release by alveolar MØ and tumor necrosis factor (TNF)-alpha and nitric oxide levels in the lung lavage were monitored. Normal alveolar MØ had undetectable levels of COX-2 on Western blots. However, 1 day after intranasal administration, almost all alveolar MØ had phagocytosed HK-BCG and expressed NE-dissociated COX-2 without any increase in the release of PGE(2). At 28 days after intranasal administration, 68% of alveolar MØ still contained both BCG and the NE-dissociated form of COX-2. NE-associated (active) COX-2 was not observed in alveolar MØ. In contrast, 7 days after intraperitoneal injection of HK-BCG, peritoneal MØ containing HK-BCG were no longer detected. At 28 days after intranasal administration, TNF-alpha and nitrite levels in the lung lavage fluid were significantly higher than those in controls. Our results indicate that mycobacterial pulmonary inflammation is associated with suppressed PGE(2) production by alveolar MØ, with expression of COX-2 dissociated from the NE.

Transcriptional repressor REST drives lineage stage-specific chromatin compaction at Ptch1 and increases AKT activation in a mouse model of medulloblastoma.

In medulloblastomas (MBs), the expression and activity of RE1-silencing transcription factor (REST) is increased in tumors driven by the sonic hedgehog (SHH) pathway, specifically the SHH-α (children 3 to 16 years) and SHH-ß (infants) subgroups. Neuronal maturation is greater in SHH-ß than SHH-α tumors, but both correlate with poor overall patient survival. We studied the contribution of REST to MB using a transgenic mouse model (RESTTG ) wherein conditional NeuroD2-controlled REST transgene expression in lineage-committed Ptch1 +/- cerebellar granule neuron progenitors (CGNPs) accelerated tumorigenesis and increased penetrance and infiltrative disease. This model revealed a neuronal maturation context-specific antagonistic interplay between the transcriptional repressor REST and the activator GLI1 at Ptch1 Expression of Arrb1, which encodes ß-arrestin1 (a GLI1 inhibitor), was substantially reduced in proliferating and, to a lesser extent, lineage-committed RESTTG cells compared with wild-type proliferating CGNPs. Lineage-committed RESTTG cells also had decreased GLI1 activity and increased histone H3K9 methylation at the Ptch1 locus, which correlated with premature silencing of Ptch1 These cells also had decreased expression of Pten, which encodes a negative regulator of the kinase AKT. Expression of PTCH1 and GLI1 were less, and ARRB1 was somewhat greater, in patient SHH-ß than SHH-α MBs, whereas that of PTEN was similarly lower in both subtypes than in others. Inhibition of histone modifiers or AKT reduced proliferation and induced apoptosis, respectively, in cultured REST-high MB cells. Our findings linking REST to differentiation-specific chromatin remodeling, PTCH1 silencing, and AKT activation in MB tissues reveal potential subgroup-specific therapeutic targets for MB patients.