Autophagy plays a broad role in health and disease. Here, we show that inositol polyphosphate multikinase (IPMK) is a prominent physiological determinant of autophagy and is critical for liver inflammation and regeneration. Deletion of IPMK diminishes autophagy in cell lines and mouse liver. Regulation of autophagy by IPMK does not require catalytic activity. Two signaling axes, IPMK-AMPK-Sirt-1 and IPMK-AMPK-ULK1, appear to mediate the influence of IPMK on autophagy. IPMK enhances autophagy-related transcription by stimulating AMPK-dependent Sirt-1 activation, which mediates the deacetylation of histone 4 lysine 16. Furthermore, direct binding of IPMK to ULK and AMPK forms a ternary complex that facilitates AMPK-dependent ULK phosphorylation. Deletion of IPMK in cell lines and intact mice virtually abolishes lipophagy, promotes liver damage as well as inflammation, and impairs hepatocyte regeneration. Thus, targeting IPMK may afford therapeutic benefits in disabilities that depend on autophagy and lipophagy-specifically, in liver inflammation and regeneration.

KIAA1549-BRAF fusion is the most common genetic event in pilocytic astrocytoma (PA), and leads to activation of the mitogen activated protein kinase (MAPK) signaling pathway. Fusions of BRAF with other partner genes, as well as other genetic alterations not involving BRAF but also leading to MAPK pathway activation have been described rarely.

Pediatric high-grade gliomas (pHGGs) are incurable malignant brain cancers. Clear somatic genetic drivers are difficult to identify in the majority of cases. We hypothesized that this may be due to the existence of germline variants that influence tumor etiology and/or progression and are filtered out using traditional pipelines for somatic mutation calling.

T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants. T cells electroporated with nucleoside-modified and purified mRNA encoding CD19 CAR showed an initial twofold increase in CAR surface expression, as well as a twofold improvement in cytotoxic killing of leukemia cells that persisted up to 5 days. T cells generated with nucleoside-modified and purified CAR mRNA also showed reduced expression of checkpoint regulators and a differential pattern of genetic activation compared to those made with conventional mRNA. In vivo studies using a leukemia mouse model revealed that the most robust 100-fold suppression of leukemic burden was achieved using T cells electroporated with purified mRNAs, regardless of their nucleoside modification. The results provide a novel approach to generate mRNA for clinical trials, and poise mRNA CAR T cells for increased efficacy during testing as new CAR targets emerge.

Colon cancer recurrence after therapy, such as 5-fluorouracil (5-FU), remains a challenge in the clinical setting. Chemotherapy reduces tumor burden by inducing cell death; however, the resulting dead tumor cells, or debris, may paradoxically stimulate angiogenesis, inflammation, and tumor growth. Here, we demonstrate that 5-FU-generated colon carcinoma debris stimulates the growth of a subthreshold inoculum of living tumor cells in subcutaneous and orthotopic models. Debris triggered the release of osteopontin (OPN) by tumor cells and host macrophages. Both coinjection of debris and systemic treatment with 5-FU increased plasma OPN levels in tumor-bearing mice. RNA expression levels of secreted phosphoprotein 1, the gene that encodes OPN, correlate with poor prognosis in patients with colorectal cancer and are elevated in chemotherapy-treated patients who experience tumor recurrence vs. no recurrence. Pharmacologic and genetic ablation of OPN inhibited debris-stimulated tumor growth. Systemic treatment with a combination of a neutralizing OPN antibody and 5-FU dramatically inhibited tumor growth. These results demonstrate a novel mechanism of tumor progression mediated by OPN released in response to chemotherapy-generated tumor cell debris. Neutralization of debris-stimulated OPN represents a potential therapeutic strategy to overcome the inherent limitation of cytotoxic therapies as a result of the generation of cell debris.-Chang, J., Bhasin, S. S., Bielenberg, D. R., Sukhatme, V. P., Bhasin, M., Huang, S., Kieran, M. W., Panigrahy, D. Chemotherapy-generated cell debris stimulates colon carcinoma tumor growth via osteopontin.

Children diagnosed with brain tumors have the lowest overall survival of all pediatric cancers. Recent molecular studies have resulted in the discovery of recurrent driver mutations in many pediatric brain tumors. However, despite these molecular advances, the clinical outcomes of high grade tumors, including H3K27M diffuse midline glioma (H3K27M DMG), remain poor. To address the paucity of tissue for biological studies, we have established a comprehensive protocol for the coordination and processing of donated specimens at postmortem. Since 2010, 60 postmortem pediatric brain tumor donations from 26 institutions were coordinated and collected. Patient derived xenograft models and cell cultures were successfully created (76% and 44% of attempts respectively), irrespective of postmortem processing time. Histological analysis of mid-sagittal whole brain sections revealed evidence of treatment response, immune cell infiltration and the migratory path of infiltrating H3K27M DMG cells into other midline structures and cerebral lobes. Sequencing of primary and disseminated tumors confirmed the presence of oncogenic driver mutations and their obligate partners. Our findings highlight the importance of postmortem tissue donations as an invaluable resource to accelerate research, potentially leading to improved outcomes for children with aggressive brain tumors.

How abnormal neurodevelopment relates to the tumour aggressiveness of medulloblastoma (MB), the most common type of embryonal tumour, remains elusive. Here we uncover a neurodevelopmental epigenomic programme that is hijacked to induce MB metastatic dissemination. Unsupervised analyses of integrated publicly available datasets with our newly generated data reveal that SMARCD3 (also known as BAF60C) regulates Disabled 1 (DAB1)-mediated Reelin signalling in Purkinje cell migration and MB metastasis by orchestrating cis-regulatory elements at the DAB1 locus. We further identify that a core set of transcription factors, enhancer of zeste homologue 2 (EZH2) and nuclear factor I X (NFIX), coordinates with the cis-regulatory elements at the SMARCD3 locus to form a chromatin hub to control SMARCD3 expression in the developing cerebellum and in metastatic MB. Increased SMARCD3 expression activates Reelin-DAB1-mediated Src kinase signalling, which results in a MB response to Src inhibition. These data deepen our understanding of how neurodevelopmental programming influences disease progression and provide a potential therapeutic option for patients with MB.

With the increasing rates of exome and whole genome sequencing, the ability to classify large sets of germline sequencing variants using up-to-date American College of Medical Genetics - Association for Molecular Pathology (ACMG-AMP) criteria is crucial. Here, we present Automated Germline Variant Pathogenicity (AutoGVP), a tool that integrates germline variant pathogenicity annotations from ClinVar and sequence variant classifications from a modified version of InterVar (PVS1 strength adjustments, removal of PP5/BP6). This tool facilitates large-scale, clinically-focused classification of germline sequence variants in a research setting.

Craniopharyngiomas are epithelial tumors that typically arise in the suprasellar region of the brain. Patients experience substantial clinical sequelae from both extension of the tumors and therapeutic interventions that damage the optic chiasm, the pituitary stalk and the hypothalamic area. Using whole-exome sequencing, we identified mutations in CTNNB1 (β-catenin) in nearly all adamantinomatous craniopharyngiomas examined (11/12, 92%) and recurrent mutations in BRAF (resulting in p.Val600Glu) in all papillary craniopharyngiomas (3/3, 100%). Targeted genotyping revealed BRAF p.Val600Glu in 95% of papillary craniopharyngiomas (36 of 39 tumors) and mutation of CTNNB1 in 96% of adamantinomatous craniopharyngiomas (51 of 53 tumors). The CTNNB1 and BRAF mutations were clonal in each tumor subtype, and we detected no other recurrent mutations or genomic aberrations in either subtype. Adamantinomatous and papillary craniopharyngiomas harbor mutations that are mutually exclusive and clonal. These findings have important implications for the diagnosis and treatment of these neoplasms.

Activation of the RAS-RAF-MEK-ERK signaling pathway is thought to be the key driver of pediatric low-grade astrocytoma (PLGA) growth. Sorafenib is a multikinase inhibitor targeting BRAF, VEGFR, PDGFR, and c-kit. This multicenter phase II study was conducted to determine the response rate to sorafenib in patients with recurrent or progressive PLGA.

Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.

The HERBY trial was a phase II open-label, randomized, multicenter trial evaluating bevacizumab (BEV) in addition to temozolomide/radiotherapy in patients with newly diagnosed non-brainstem high-grade glioma (HGG) between the ages of 3 and 18 years. We carried out comprehensive molecular analysis integrated with pathology, radiology, and immune profiling. In post-hoc subgroup analysis, hypermutator tumors (mismatch repair deficiency and somatic POLE/POLD1 mutations) and those biologically resembling pleomorphic xanthoastrocytoma ([PXA]-like, driven by BRAF_V600E or NF1 mutation) had significantly more CD8+ tumor-infiltrating lymphocytes, and longer survival with the addition of BEV. Histone H3 subgroups (hemispheric G34R/V and midline K27M) had a worse outcome and were immune cold. Future clinical trials will need to take into account the diversity represented by the term "HGG" in the pediatric population.

We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection.

Angiocentric gliomas are pediatric low-grade gliomas (PLGGs) without known recurrent genetic drivers. We performed genomic analysis of new and published data from 249 PLGGs, including 19 angiocentric gliomas. We identified MYB-QKI fusions as a specific and single candidate driver event in angiocentric gliomas. In vitro and in vivo functional studies show that MYB-QKI rearrangements promote tumorigenesis through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression and hemizygous loss of the tumor suppressor QKI. To our knowledge, this represents the first example of a single driver rearrangement simultaneously transforming cells via three genetic and epigenetic mechanisms in a tumor.

The chromodomain, helicase DNA-binding protein 5 (CHD5) is a potential tumor suppressor gene located on chromosome 1p36, a region recurrently deleted in high risk neuroblastoma (NB). Previous data have shown that CHD5 mRNA is present in normal neural tissues and in low risk NB, nevertheless, the distribution of CHD5 protein has not been explored. The aim of this study was to investigate CHD5 protein expression as an immunohistochemical marker of outcome in NB. With this purpose, CHD5 protein expression was analyzed in normal neural tissues and neuroblastic tumors (NTs). CHD5 gene and protein expression was reexamined after induction chemotherapy in a subset of high risk tumors to identify potential changes reflecting tumor response.

Dramatic advances in the molecular analysis of diffuse intrinsic pontine glioma have occurred over the last decade and resulted in the identification of potential therapeutic targets. In spite of these advances, no significant improvement in the outcome has been achieved and median survival remains approximately 10 months. An understanding of the approaches that have been taken to date, why they failed, and how that information can lead the field forward is critical if we are to change the status quo. In this review, we will discuss the clinical trial landscape in North America with an overview of historical approaches that failed and what might account for this failure. We will then provide a discussion of how our understanding of the genotype of this disease has led to the development of a number of trials targeting the mutations and epigenome of diffuse intrinsic pontine gliomas and the issues related to these trials. Similarly, the introduction of methodologies to address penetration across the blood-brain barrier will be considered in the context of both targeted approaches, epigenetic modification, and immune surveillance of these tumors. The comprehensive analysis of these data, generated through cooperative groups, collaborative clinical trials, and pilot studies in North America will be the focus of the IVth Memorial Alicia Pueyo international symposium in Barcelona on March 12th, 2018 and will be compared and contrasted with a similar comprehensive analysis of the European data with the goal of bringing all of these data together to develop a uniform platform on which new rational trials can be based.

Inflammation in the tumor bed can either promote or inhibit tumor growth. Peroxisome proliferator-activated receptor (PPAR)alpha is a central transcriptional suppressor of inflammation, and may therefore modulate tumor growth. Here we show that PPARalpha deficiency in the host leads to overt inflammation that suppresses angiogenesis via excess production of the endogenous angiogenesis inhibitor thrombospondin-1 and prevents tumor growth. Bone marrow transplantation and granulocyte depletion show that PPARalpha expressing granulocytes are necessary for tumor growth. Neutralization of thrombospondin-1 restores tumor growth in PPARalpha-deficient mice. These findings suggest that the absence of PPARalpha activity renders inflammatory infiltrates tumor suppressive and, thus, may provide a target for inhibiting tumor growth by modulating stromal processes, such as angiogenesis.

Glioblastoma (GBM) is the most aggressive nervous system cancer. Understanding its molecular pathogenesis is crucial to improving diagnosis and treatment. Integrated analysis of genomic, proteomic, post-translational modification and metabolomic data on 99 treatment-naive GBMs provides insights to GBM biology. We identify key phosphorylation events (e.g., phosphorylated PTPN11 and PLCG1) as potential switches mediating oncogenic pathway activation, as well as potential targets for EGFR-, TP53-, and RB1-altered tumors. Immune subtypes with distinct immune cell types are discovered using bulk omics methodologies, validated by snRNA-seq, and correlated with specific expression and histone acetylation patterns. Histone H2B acetylation in classical-like and immune-low GBM is driven largely by BRDs, CREBBP, and EP300. Integrated metabolomic and proteomic data identify specific lipid distributions across subtypes and distinct global metabolic changes in IDH-mutated tumors. This work highlights biological relationships that could contribute to stratification of GBM patients for more effective treatment.

International cancer registries make real-world genomic and clinical data available, but their joint analysis remains a challenge. AACR Project GENIE, an international cancer registry collecting data from 19 cancer centers, makes data from >130,000 patients publicly available through the cBioPortal for Cancer Genomics (https://genie.cbioportal.org). For 25,000 patients, additional real-world longitudinal clinical data, including treatment and outcome data, are being collected by the AACR Project GENIE Biopharma Collaborative using the PRISSMM data curation model. Several thousand of these cases are now also available in cBioPortal. We have significantly enhanced the functionalities of cBioPortal to support the visualization and analysis of this rich clinico-genomic linked dataset, as well as datasets generated by other centers and consortia. Examples of these enhancements include (i) visualization of the longitudinal clinical and genomic data at the patient level, including timelines for diagnoses, treatments, and outcomes; (ii) the ability to select samples based on treatment status, facilitating a comparison of molecular and clinical attributes between samples before and after a specific treatment; and (iii) survival analysis estimates based on individual treatment regimens received. Together, these features provide cBioPortal users with a toolkit to interactively investigate complex clinico-genomic data to generate hypotheses and make discoveries about the impact of specific genomic variants on prognosis and therapeutic sensitivities in cancer.

High throughput molecular sequencing and increased biospecimen variety have introduced significant informatics challenges for research biorepository infrastructures. We applied a modular system integration approach to develop an operational biorepository management system. This method enables aggregation of the clinical, specimen and genomic data collected for biorepository resources.