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Purpose:Gliomas, a genetically heterogeneous group of primary central nervous system tumors, continue to pose a significant clinical challenge. Discovery of chromosomal rearrangements involving kinase genes has enabled precision therapy, and improved outcomes in several malignancies.Experimental Design:Positing that similar benefit could be accomplished for patients with brain cancer, we evaluated The Cancer Genome Atlas (TCGA) glioblastoma dataset. Functional validation of the oncogenic potential and inhibitory sensitivity of discovered ROS1 fusions was performed using three independent cell-based model systems, and an in vivo murine xenograft study.Results:In silico analysis revealed previously unreported intrachromosomal 6q22 microdeletions that generate ROS1-fusions from TCGA glioblastoma dataset. ROS1 fusions in primary glioma and ependymoma were independently corroborated from MSK-IMPACT and Foundation Medicine clinical datasets. GOPC–ROS1 is a recurrent ROS1 fusion in primary central nervous system (CNS) tumors. CEP85L–ROS1 and GOPC–ROS1 are transforming oncogenes in cells of astrocytic lineage, and amenable to pharmacologic inhibition with several ROS1 inhibitors even when occurring concurrently with other cancer hotspot aberrations frequently associated with glioblastoma. Oral monotherapy with a brain-permeable ROS1 inhibitor, lorlatinib, significantly prolonged survival in an intracranially xenografted tumor model generated from a ROS1 fusion-positive glioblastoma cell line.Conclusions:Our findings highlight that CNS tumors should be specifically interrogated for these rare intrachromosomal 6q22 microdeletion events that generate actionable ROS1 fusions. ROS1 fusions in primary brain cancer may be amenable for clinical intervention with kinase inhibitors, and this holds the potential of novel treatment paradigms in these treatment-refractory cancer types, particularly in glioblastoma.

Supplementary Tables 1-11

U118MG spheroid treated with 250nM lorlatinib for 72 hrs

Supplementary Figures 1-5

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.

Supplementary Figures

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-β 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.

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. Here we show 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-β receptor agonist (LTβRag) 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 identify p53 as a key regulator of immune evasion, and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.

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.

Many immunotherapies act by enhancing the ability of T cells to kill tumor cells. But T cell killing depends on recognition of antigens presented by Class I MHC (MHC-I) on the tumor cell surface; if a tumor lacks MHC-I, it cannot be recognized by T cells. To study the efficacy of immunotherapy for Group 3 medulloblastoma, we have used mouse models driven by overexpression of Myc and dominant negative p53 (“MP tumors”), or by overexpression of Myc and Gfi1 (“MG tumors”). While MP tumors grow in immunocompetent mice, MG tumors don’t grow, due to T-cell rejection. To understand this difference, we analyzed expression of immunoregulatory molecules, and found that MP tumors completely lack surface MHC-I. Mechanistically, this is because two key proteins required for MHC-I trafficking – TAP1 and ERAP1 – are targets of p53; since MP tumors lack functional p53, they also lack TAP1 and ERAP1, and surface MHC-I. These studies suggest that p53 plays a critical role in determining immunogenicity, and that p53-mutant tumors are resistant to immune attack. To overcome this resistance, we treated tumor cells with agents that regulate MHC-1. We found that low doses of tumor necrosis factor alpha (TNF) can rescue expression of ERAP, TAP and MHC-I on MP tumor cells. In vivo, TNF prolongs survival of tumor-bearing mice, and markedly augments the efficacy of immune checkpoint inhibitors. These results raise the possibility that TNF could be used prior to immunotherapy to render tumors more sensitive to T cell attack.

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-β 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.

Many immunotherapies act by enhancing T-cell killing of tumor cells. Cytotoxic T cells recognize antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. Our studies suggest that medulloblastomas and high-grade gliomas 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. Treatment with tumor necrosis factor or lymphotoxin beta receptor agonist rescues expression of Erap1, Tap1, and MHC-I on p53 mutant tumor cells. In vivo, TNF treatment prolongs survival and markedly augments the efficacy of the immune checkpoint inhibitor anti-PD-1. These studies identify p53 as a key regulator of immune evasion in vivo and suggest that TNF could be used to enhance sensitivity of p53-mutant tumors to immunotherapy. Citation Format: Alexandra Garancher, Hiromichi Suzuki, Svasti Haricharan, Meher B. Masihi, Jessica M. Rusert, Paula S. Norris, Florent Carrette, Megan M. Romero, Sorana A. Morrissy, Patryk Skowron, Florence M.G. Cavalli, Hamza Farooq, Vijay Ramaswamy, Alaide Morcavallo, Jacob J. Henderson, James M. Olson, Yoon-Jae Cho, Xiao-Nan Li, Louis Chesler, Marco A. Marra, Oren J. Becher, Linda M. Bradley, Carl F. Ware, Michael D. Taylor, Robert J. Wechsler-Reya. Overcoming immune evasion in pediatric brain tumors [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA11.

Purpose: Gliomas, a genetically heterogeneous group of primary central nervous system tumors, continue to pose a significant clinical challenge. Discovery of chromosomal rearrangements involving kinase genes has enabled precision therapy, and improved outcomes in several malignancies. Experimental Design: Positing that similar benefit could be accomplished for patients with brain cancer, we evaluated The Cancer Genome Atlas (TCGA) glioblastoma dataset. Functional validation of the oncogenic potential and inhibitory sensitivity of discovered ROS1 fusions was performed using three independent cell-based model systems, and an in vivo murine xenograft study. Results: In silico analysis revealed previously unreported intrachromosomal 6q22 microdeletions that generate ROS1-fusions from TCGA glioblastoma dataset. ROS1 fusions in primary glioma and ependymoma were independently corroborated from MSK-IMPACT and Foundation Medicine clinical datasets. GOPC–ROS1 is a recurrent ROS1 fusion in primary central nervous system (CNS) tumors. CEP85L–ROS1 and GOPC–ROS1 are transforming oncogenes in cells of astrocytic lineage, and amenable to pharmacologic inhibition with several ROS1 inhibitors even when occurring concurrently with other cancer hotspot aberrations frequently associated with glioblastoma. Oral monotherapy with a brain-permeable ROS1 inhibitor, lorlatinib, significantly prolonged survival in an intracranially xenografted tumor model generated from a ROS1 fusion-positive glioblastoma cell line. Conclusions: Our findings highlight that CNS tumors should be specifically interrogated for these rare intrachromosomal 6q22 microdeletion events that generate actionable ROS1 fusions. ROS1 fusions in primary brain cancer may be amenable for clinical intervention with kinase inhibitors, and this holds the potential of novel treatment paradigms in these treatment-refractory cancer types, particularly in glioblastoma.

Group 3, MYC-amplified medulloblastomas (MB) remain exceedingly difficult to treat despite maximum tolerable doses of chemotherapy and radiation. Additionally, strategies to directly target MYC are not readily available for clinical use in pediatrics. Thus, to identify novel genetic dependencies and potential therapeutic targets in MYC-amplified MB tumors, we screened MB cell lines using a high-throughput shRNA library of 102,000 unique hairpins targeting nearly all genes in the coding genome. As expected, we observed genes such as MYC and components of the PI3K and FOXO pathways as statistically significant genetic dependencies in MYC-amplified MB cells relative to >70 non-MB cancer cell lines. However, we also observed consistent and marked sensitivity to hairpins targeting genes in the TGFb pathway. Supporting these findings, a meta-analysis of multiple genomic profiling studies showed mutations and/or copy number variations in several components of the TGFb pathway in Group 3 MBs and we further confirmed the presence of activated TGFb/SMAD pathway in Group 3/MYC-amplified MB tumors. Treatment of MYC-amplified MB cells with the FDA-approved TGFb inhibitor, LY-2157299, resulted in marked inhibition of phospho-SMAD2/3 levels as well as inhibition of non-canonical targets of the TGFb pathway. Proliferation assays showed LY-2157299 preferentially inhibited growth in MYC-amplified MB cells compared to non-MYC-amplified MB lines. This growth inhibition was accompanied by loss of markers of epithelial to mesenchymal transition, a known proxy for infiltrative and metastatic phenotype. Lastly, mice harboring patient-derived orthotopic xenografts treated with LY-2157299 lived significantly longer relative to vehicle-treated mice with immunohistochemical confirmation of downregulated phospho-SMAD2 and increased cleaved caspase, in vivo. Thus, our functional genomic and pre-clinical validation studies nominate TGFb pathway inhibitors as a therapeutic strategy for patients with Group 3/MYC-amplified medulloblastomas.

Many immunotherapies act by enhancing T cell killing of tumor cells. CD8+ cytotoxic T cells recognize antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. Here we show 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. Treatment with tumor necrosis factor (TNF) or lymphotoxin beta receptor agonist (LTβRag) rescues expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, TNF treatment prolongs survival and markedly augments the efficacy of the immune checkpoint inhibitor anti-PD-1. These studies identify p53 as a key regulator of immune evasion in vivo, and suggest that TNF could be used to enhance sensitivity of p53-mutant tumors to immunotherapy.

Group 3 medulloblastomas (MB) harboring MYC gene amplification are generally considered a relative immune desert. This is despite often carrying a relatively high mutational burden, which is associated with immune checkpoint inhibitor sensitivity in other cancers. For this reason the use of checkpoint inhibitors has largely been dismissed as a potential treatment strategy in these high risk tumors. However, our work using TGFb receptor small molecule inhibitors (TGFbRi) in MYC-amplified MB has shown that inhibiting the TGFb pathway results in increased antigen presenting machinery and checkpoint ligands. And while TGFb pathway inhibition alone significantly prolongs survival in MB-bearing mice, it was not curative with all animals eventually dying of disease. Therefore, we set out to exploit the tumor intrinsic and extrinsic changes that occur in MB with TGFb pathway inhibition and hypothesized that combining TGFb pathway inhibition with immune checkpoint inhibition would result in meaningful improvements in rates of survival. Indeed, “priming” MBs with a TGFbRi in conjunction with weekly administration of OX40 agonist resulted in tumor rejection and durable overall survival relative to either therapy alone. Intracranial tumors collected at various time points throughout TGFbRi “priming” showed increased cell surface expression of OX40, PD-L1 and MHC class one molecules within 1–2 days of TGFbRi treatment. Concurrently, there was a consistent increase in CD4+ and CD8+ T cells into the tumor, which was potentiated with OX40 agonism. These findings imply that the sequence of TGFb receptor inhibition followed by OX40 agonism could be a potent, synergistic combination that should be actively explored in upcoming clinical trials. LOW GRADE GLIOMA

Developing authentic mouse models of medulloblastoma (MB) is vitally important for understanding MB biology and developing novel treatments for patients with this disease. In particular, models with a native immune system are critical for deciphering the complex roles that immune escape and surveillance play in MB formation and eradication, respectively. We previously reported that components of the TGFb signaling pathway are necessary for Group 3 MYC-amplified MB proliferation and survival. We have also observed that small molecule inhibition of the TGFb pathway primes MB for immune checkpoint allowing for increased T cell infiltration in an otherwise “immunodepleted” tumor. Here, we sought to determine whether TGFb signaling in cooperation with MYC was sufficient to transform cells into MB. Toward this end, we isolated cerebellar stem cells from 4–6 week old mice and transduced them with lentiviral vectors expressing either a constitutively active TGFbR1 (ALK5) or MYC, or both. Cells transduced with both ALK5 and MYC, but not each individually, quickly formed dense neurospheres in vitro that propagated ad infinitum. When allografted into either immunodeficient or syngeneic immune competent mice, these transformed cells formed large, infiltrating tumors, that displayed nuclear atypia and other features reminiscent of anaplastic MB. Gene expression microarray analysis further confirmed an expression signature consistent with previously published Group 3 MYC-amplified MB mouse models (GTML; MYC-DNp53) as well as human primary Group 3 MB. Interestingly, tumors generated from TGFb/MYC-transformed cells allografted into syngeneic immune competent mice lacked T cell infiltration and had dysfunctional neoantigen presenting machinery consistent with the human condition. Thus, we provide a novel Group 3 TGFb/MYC-driven MB model that closely recapitulates the human condition and will serve as a useful resource to better understand mechanisms of tumor-immune interactions in MB. SIGNALING

Embryonal tumors (ET) of the central nervous system (CNS) in children encompass a wide clinical spectrum of aggressive malignancies. Until recently, the overlapping morphological features of these lesions posed a diagnostic challenge and undermined discovery of optimal treatment strategies. However, with the advances in genomic technology and the outpouring of biological data over the last decade, clear insights into the molecular heterogeneity of these tumors are now well delineated. The major subtypes of ETs of the CNS in children include medulloblastoma, atypical teratoid rhabdoid tumor (ATRT), and embryonal tumors with multilayered rosettes (ETMR), which are now biologically and clinically characterized as different entities. These important developments have paved the way for treatments guided by risk stratification as well as novel targeted therapies in efforts to improve survival and reduce treatment burden.

Medulloblastoma is the most common malignant brain tumor of childhood. To identify targetable vulnerabilities, we employed inhibitor screening that revealed mTOR inhibitor hypersensitivity in the MYC-overexpressing medulloblastoma cell line, D341. Concomitant exome sequencing unveiled an uncharacterized missense mutation, TSC2A415V , in these cells. We biochemically demonstrate that the TSC2A415V mutation is functionally deleterious, leading to shortened half-life and proteasome-mediated protein degradation. These data suggest that MYC cooperates with activated kinase pathways, enabling pharmacologic intervention in these treatment refractory tumors. We propose that identification of activated kinase pathways may allow for tailoring targeted therapy to improve survival and treatment-related morbidity in medulloblastoma.