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Purpose: Anaplastic Lymphoma Kinase (ALK) aberrations have been identified in pediatric type infant gliomas, but their occurrence across age groups, functional effects, and treatment response have not been broadly established. Experimental Design: We performed a comprehensive analysis of ALK expression and genomic aberrations in both newly-generated and retrospective data from 371 glioblastomas (156 adult, 205 infant/pediatric and 10 congenital) with in vitro and in vivo validation of aberrations. Results: ALK aberrations at the protein or genomic level were detected in 12% of gliomas (45/371) in a wide age range (0-80 years). Recurrent as well as novel ALK fusions (LRRFIP1-ALK, DCTN1-ALK, PRKD3-ALK) were present in 50% (5/10) of congenital/infant, 1.4% (3/205) of pediatric, and 1.9% (3/156) of adult GBMs. ALK fusions were present as the only candidate driver in congenital/infant GBMs, and were sometimes focally amplified. In contrast, adult ALK fusions co-occurred with other oncogenic drivers. No activating ALK mutations were identified in any age group. Novel and recurrent ALK rearrangements promoted STAT3 and ERK1/2 pathways and transformation in vitro and in vivo. ALK-fused GBM cellular and mouse models were responsive to ALK inhibitors, including in patient cells derived from a congenital GBM. Relevant to treatment of infant gliomas, we showed that ALK protein appears minimally expressed in the forebrain at perinatal stages and no gross effects on perinatal brain development was seen in pregnant mice treated with the ALK inhibitor ceritinib. Conclusions: These findings support expanded evaluation of brain-penetrant ALK inhibitors in clinical trials across infant, pediatric, and adult GBMs. Citation Format: Anne-Florence Blandin, Ross Giglio, Maya Srikanth Graham, Guadalupe Garcia, Seth Malinowski, Jared K. Woods, Shakti Ramkissoon, Lori Ramkissoon, Frank Dubois, Kate Schoolcraft, Jessica W. Tsai, Dayle K. Wang, Robert Jones, Jayne Vogelzang, Kristine Pelton, Sarah Becker, Fiona Watkinson, Claire Sinai, Elizabeth Cohen, Matthew Booker, Michael Tolstorukov, Veerle Haemels, Liliana Goumnerova, Karen Wright, Mark Kieran, Katie Fehnel, David Reardon, Arnault Tauziede-Espariat, Rishi Lulla, Benjamin Carcamo, Stanley Chaleff, Alain Charest, Frederik De Smet, Azra H. Ligon, Adrian Dubuc, Melanie Pagès, Pascale Varlet, Patrick Wen, Brian Alexander, Susan Chi, Sanda Alexandrescu, Ralf Kittler, Robert Bachoo, Rameen Beroukhim, Pratiti Bandopadhayay, Keith L. Ligon. ALK amplification and rearrangements are recurrent targetable events in congenital and adult glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1201.

Background: Diffuse midline gliomas (DMGs) are a universally fatal brain tumor of childhood. While histone mutations are a critical tumor initiating event, they are insufficient to drive gliomagenesis. Histone mutations co-occur with somatic alterations in other pathways including TP53, MAPK, and MYC signaling. However, the mechanisms through which these pathways are activated have not been fully elucidated. Methods: We applied an integrative approach using transcriptomics, epigenetics, proteomics, in vitro cancer models, and in vivo mouse models to systematically evaluate how FOXR2 mediates gliomagenesis. Results: We have recently found that a subset of DMGs aberrantly express FOXR2, a forkhead transcription factor. FOXR2 is both sufficient to enhance tumor formation, and necessary for FOXR2-expressing DMGs. While FOXR2 indeed enhances MYC protein stability, FOXR2 exerts oncogenesis through MYC-independent functions and specifically hijacks E26-transformation specific (ETS) transcriptional circuits and FOXR2 DNA-binding is highly enriched at ETS motifs. We have performed proteomic and phospho-proteomic analysis of FOXR2-expressing human neural stem cells to identify proteins and phospho-sites that are highly enriched in FOXR2-expressing cells. Conclusion: Taken together, this study elucidates how FOXR2 interacts with ETS transcription factors to mediate oncogenesis, and further highlights a role for FOXR2 in activating ETS and MAPK signaling. Citation Format: Jessica W. Tsai, Paloma Cejas, Marissa Coppola, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Annarah Charles, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Volker Hovestadt, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. Dissecting mechanisms underlying FOXR2-mediated gliomagenesis in diffuse midline gliomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3562.

PDGFRA has been shown to be commonly altered in high-grade gliomas (HGGs), including histone 3 lysine 27-mutated diffuse midline gliomas (H3K27M DMG), a disease with almost no long-term survivors. Here, we performed comprehensive genomic and transcriptomic analysis of 260 high-grade glioma cases, which revealed PDGFRA genomic alterations (mutations and/or amplifications) in 13% of patients. H3K27M DMGs had significantly higher PDGFRA expression compared to H3 wild-type tumors, and PDGFRA gene amplification resulted in even higher expression levels in H3K27M DMGs as well as H3 wild-type HGGs. We tested a panel of patient- derived pHGG/H3K27M DMG models against a range of PDGFRA inhibitors, including avapritinib, a potent small molecule inhibitor with relatively selective activity against both wild-type and mutant PDGFRA. Avapritinib showed supra-micromolar blood-brain barrier penetration in our pre-clinical models and demonstrated significant survival impact in an aggressive patient-derived H3K27M DMG mouse xenograft model. Finally, building on this preclinical activity, we report here the first clinical experience using avapritinib in eight pediatric and young adult patients with high-grade glioma (H3K27M DMG and/or PDGFRA altered). Avapritinib has thus far been well tolerated with no significant acute toxicities. Most importantly, our preliminary data reveal radiographic response evaluated by RAPNO criteria in 50% of patients, a striking outcome rarely seen in this patient population. In summary, we report that avapritinib is a selective, CNS-penetrant small molecule inhibitor of PDGFRA that shows potent activity in preclinical models and produces promising clinical responses with good tolerability in patients with high-grade glioma. This suggests a promising role for avapritinib therapy in this population with previously dismal outcomes. Citation Format: Lisa Mayr, Maria Trissal, Kallen Schwark, Jenna Labelle, Andrew Groves, Julia Furtner-Srajer, Jeffrey Supko, Liesa Weiler-Wichtl, Olivia Hack, Jacob Rozowsky, Joana G. Marques, Eshini Pandatharatna, Ulrike Leiss, Verena Rosenmayr, Frank Dubois, Noah F. Greenwald, Sibylle Madlener, Armin S. Guntner, Hana Pálová, Natalia Stepien, Daniela Lötsch-Gojo, Christian Dorfer, Karin Dieckmann, Andreas Peyrl, Amedeo A. Azizi, Alicia Baumgartner, Ondřej Slabý, Petra Pokorná, Pratiti Bandopadhayay, Rameen Beroukhim, Keith Ligon, Christof Kramm, Annika Bronsema, Simon Bailey, Ana Guerreiro Stücklin, Sabine Mueller, David T. Jones, Natalie Jäger, Jaroslav Štěrba, Leonhard Müllauer, Christine Haberler, Chandan Kumar-Sinha, Arul Chinnaiyan, Rajen Mody, Mary Skrypek, Nina Martinez, Daniel C. Bowers, Carl Koschmann, Johannes Gojo, Mariella Filbin. Clinical response to the PDGFRα inhibitor avapritinib in high-grade glioma patients. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5719.

Pediatric pan-cancer genome analyses do not capture the full range of diagnoses encountered in clinical practice. To inform basket trial design and real-world precision oncology practice, we classified diagnoses and assessed the landscape of mutations, including trial-matching, in an unselected cohort of pediatric solid tumors. Since 2013 all Dana-Farber/Boston Children’s patients have been offered participation in the Profile study. Participant tumor samples were sequenced with DFCI-OncoPanel, a targeted panel test sequencing exons of up to 447 cancer genes for single nucleotide variants, insertions and deletions and copy number alterations, and introns and exons of up to 60 genes for rearrangements. Patient diagnosis was classified according to ICD-O, version 3.2. Genomic alterations were analyzed for matching to the actionable mutation lists of precision oncology basket trials (NCI-COG Pediatric MATCH, NCI-MATCH, and the ASCO TAPUR Study v.3). Data will be shared with the Childhood Cancer Data Initiative. There were 888 pediatric patients with sequencing enrolled in Profile between January 2013 and March 2019; 512 (58%) with solid tumors and 376 (42%) with CNS tumors. Fifty-five percent (491/888) of patients had one of ten common pediatric cancer diagnoses: neuroblastoma (n=80), low-grade glioma (n=72), Wilms tumor (n=57), medulloblastoma (n=55), pilocytic astrocytoma (n=47), rhabdomyosarcoma (n=44), osteosarcoma (n=42), ependymoma (n=39), Ewing sarcoma (n=28) and glioblastoma (n=27). The remaining 45% (397/888) had one of 85 distinct rare malignancies with less than 25 cases per diagnosis. Most (80/85) of these rare diagnoses are not represented in prior pediatric pan-cancer sequencing studies. Recurrent (>5%) pathogenic alterations were, in common and rare diagnoses, TP53 mutations(m) and deletions(del) and BRAFm and rearrangements(r), in common diagnoses, MYC/MYCN amplification (amp) and EWSR1r and, in rare diagnoses, CTNNB1m, CDKN2A/Bdel and NF1m/del. We found that 31% (n=271/888) of patients had at least 1 variant matching a basket trial treatment arm. Genes with matching alterations include BRAF (10%), NF1 (4%), PI3KCA (3%), NRAS (2%), BRCA2 (2%), ALK (1%), and FGFR1 (1%). Sequencing of pediatric malignancies is increasing. This study highlights opportunities to use the resulting genomic data to inform genome-selected clinical trial design and uncover drivers in pediatric cancers. The proportion of cases in this cohort with genomic alterations meeting eligibility for basket trials is equivalent to that seen in the pediatric MATCH screening study. Due to the low prevalence of the diagnoses in the long tail of cancer types in this study, defining the genomic landscape of ultra-rare cancers will require data sharing. Classifying pediatric cancer diagnoses using the ICD-O standard ontology system is feasible and will facilitate data sharing. Citation Format: Suzanne J. Forrest, Hersh Gupta, Abigail Ward, Yvonne Li, Duong Doan, Alyaa Al-Ibraheemi, Sanda Alexandrescu, Pratiti Bandopadhayay, Suzanne Shusterman, Elizabeth A. Mullen, Natalie Collins, Susan N. Chi, Karen D. Wright, Priti Kumari, Tali Mazor, Keith L. Ligon, Priyanka Shivdasani, Phani Davineni, Monica Manam, Richard L. Schilsky, Suanna S. Bruinooge, Jaime M. Guidry Auvil, Ethan Cerami, Barrett J. Rollins, Matthew L. Meyerson, Neal I. Lindeman, Laura MacConaill, Bruce E. Johnson, Andrew D. Cherniack, Alanna J. Church, Katherine A. Janeway. Sequencing of 888 pediatric solid tumors informs precision oncology trial design and data sharing initiatives in pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3890.

In the 20 years since the completion of the Human Genome Project, cancer biology remains rooted in the assumption that the human genome encodes ~20,000 protein-coding genes. Yet, I and others have shown that thousands of “non-canonical” open reading frames (ncORFs) populate the human genome, potentially representing a dramatic expansion of the cancer proteome. Despite their abundance, little is known about the role of ncORFs as cancer driver genes. We developed functional genomics approaches to pursue this question across human cancers. To determine whether ncORFs represent biologically active proteins, we experimentally interrogated 553 candidates selected from ncORF datasets. Of these, 257 (46%) showed evidence of stable protein expression using multiple assays, and 401 (72%) induced gene expression changes when expressed in cancer cell lines. The bioactivity of ncORFs was dependent on their ability to translate a protein: mutation of the ORF start codon prevented induction of gene expression changes observed with the wild type ncORF in 48 of 51 (94%) cases. Using custom CRISPR/Cas9 knock-out screens targeting >2,000 ncORFs in 20 cancer cell lines, we found that genomic knock-out of approximately 10% of ncORFs induced viability defects in cancer cells. We focused on two candidates for functional studies. In breast cancer, we described G029442 - renamed glycine-rich extracellular protein-1 (GREP1) - as a secreted protein that is highly expressed and prognostic for poor patient outcomes. Knock-out of GREP1 in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. In medulloblastoma, we found that MYC-driven medulloblastoma cells are enriched for bioactive upstream ORFs (uORFs) that are encoded within the 5’ untranslated regions of mRNAs. We validated the ASNSD1 uORF as a top genetic vulnerability in multiple models of medulloblastoma, and its overexpression is sufficient to increase neural stem cell proliferation. Mechanistically, ASNSD1 uORF promotes a MYC-associated cellular program and interacts with the multiprotein prefoldin complex, which is required for tumors to maintain post-transcriptional regulation. Our work supports a generalizable principle that ncORFs commonly encode biologically-active proteins in diverse malignancies. Ongoing investigation of ncORFs therefore represents a new frontier in cancer research with the potential to define the next generation of therapeutic target genes. Citation Format: John R. Prensner, Ian Yannuzzi, Karl Clauser, Karsten Krug, Oana Enache, Adam Brown, Amy Goodale, David E. Root, Pratiti Bandopadhayay, Todd Golub. Non-canonical proteins are cancer cell vulnerabilities in diverse malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3624.

Background: Understanding how aberrant transcription factors (TFs) hijack normal development to induce oncogenesis is a critical question in oncology. Forkhead box (FOX) proteins are a superfamily of transcriptional regulators characterized by a forkhead DNA-binding domain. Within this family, Forkhead Box R2 (FOXR2) expression has been associated with a subset of cancers including CNS and peripheral neuroblastoma. While FOXR2 has been shown to stabilize MYC isoforms, the mechanistic details through which it enhances tumor formation and the true extent of its role as an oncogene across all cancers have not been systematically evaluated. Methods: We applied an integrative approach using transcriptomics, epigenetics, in vitro cancer models, and in vivo mouse models to systematically evaluate the mechanisms by which FOXR2 is activated across human cancers. Results: We performed a pan-cancer analysis of FOXR2 activation across over 10,000 adult and pediatric cancer samples, and found FOXR2 to be aberrantly upregulated in 70% of all cancer types, and 8% of all individual tumors. We identified genetic and epigenetic mechanisms that induce its expression, including hypomethylation of a novel promoter in the vast majority (78%) of FOXR2-expressing cases. We demonstrate that FOXR2 expression is both sufficient and necessary for transformation across multiple lineages, using both in vitro and in vivo models. Conclusion: Taken together, this study demonstrates the role of FOXR2 as a potent oncogene across human cancers, and highlights a novel mechanism by which its expression is activated. Citation Format: Jessica W. Tsai, Paloma Cejas, Dayle K. Wang, Smruti Patel, David W. Wu, Phonepasong Arounleut, Xin Wei, Ningxuan Zhou, Sudeepa Syamala, Frank P. Dubois, Kristine Pelton, Jayne Vogelzang, Cecilia Sousa, Audrey Baguette, Xiaolong Chen, Alexandra L. Condurat, Sarah E. Dixon-Clarke, Kevin N. Zhou, Sophie D. Lu, Elizabeth M. Gonzalez, Madison S. Chacon, Jeromy J. Digiacomo, Rushil Kumbhani, Dana Novikov, J'Ya Hunter, Maria Tsoli, David S. Ziegler, Uta Dirksen, Natalie Jager, Gnana Prakash Balasubramanian, Christof M. Kramm, Michaela Nathrath, Stefan Bielack, Suzanne J. Baker, Jinghui Zhang, James M. McFarland, Gad Getz, Francois Aguet, Nada Jabado, Olaf Witt, Stefan M. Pfister, Keith L. Ligon, Claudia Kleinman, Henry Long, David T. Jones, Pratiti Bandopadhayay, Timothy N. Phoenix. FOXR2 is an oncogenic driver across adult and pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5730.

INTRODUCTION: Extracellular purinergic signaling plays critical roles in the regulation of tumor growth and anti-tumor immunity via autocrine/paracrine binding of metabolites to receptors on neoplastic and non-neoplastic populations. Extracellular purine concentrations are principally mediated by the ectonucleotidase enzymes CD39 and CD73, which catabolize ATP to adenosine. Within the tumor microenvironment, neoplastic, immune, and stromal cells expressing these enzymes may co-localize to generate an immunosuppressive adenosine-rich niche. However, the cellular composition, spatial architecture and phenotypic properties of these tumor ecosystems and their relationship to tumor genotype have been poorly characterized. METHODS: We quantified CD73 expression by immunohistochemistry (IHC) in a cohort of CNS tumors [meningiomas(N=222), gliomas(N=244), ependymomas(N=44), medulloblastomas(N=24), craniopharyngiomas(N=38)]. We used publicly-available single-cell RNA-seq data and 36 marker multiplexed tissue imaging (t-CyCIF) of 139 clinically and genomically annotated glioblastomas to characterize CD39 and CD73 expressing populations, define immune architecture and tumor cell states at single cell resolution, evaluate spatial correlations, and identify markers of clinical outcome. Mass spectrometry imaging (MALDI-MSI) was employed to generate spatially-resolved quantification of purine metabolite levels in glioblastoma resections (N=9). RESULTS: IHC revealed strong CD73 expression in meningiomas and gliomas. Tumor CD73 expression was associated with poor progression-free-survival in IDH-wildtype glioblastoma (p=0.04). scRNA-seq in glioblastoma revealed that CD73 is predominantly expressed by tumor cell populations, while CD39 is predominantly expressed by monocytic (macrophage, microglial) populations. t-CyCIF showed enrichment of EGFR, Ki-67, and TP53 expression in CD73-high tumor cells at a single cell level independent of genotype, as well as significant spatial correlation between CD73 expression in tumor cells and CD39 expression in macrophages. MALDI-MSI showed significantly greater adenosine concentrations in glioblastomas with high CD73 expression. CD73 expression significantly correlated with EGFR amplification or C-terminal deletion (EGFRvIII or variants), type-II interferon signaling, and PD-L1 expression in glioblastoma. CONCLUSIONS: Phenogenomic analysis of purinergic signaling in glioblastoma revealed correlations between CD73 expression and genotype, adenosine concentration, and clinical outcome. Spatial analysis revealed interaction between macrophages CD39 expression and tumor cell CD73 expression, suggesting that these populations may interact to suppress anti-tumor immunity. Anti-CD73 therapy may provide therapeutic benefits in glioblastoma by blunting immunosuppressive and oncogenic adenosine signaling. Citation Format: Shannon Coy, Jia-Ren Lin, Shu Wang, Sylwia Stopka, Rumana Rashid, Jaeho Hwang, Prasidda Khadka, Philipp Euskirchen, Pratiti Bandopadhayay, Patrick Y. Wen, Peter K. Sorger, Nathalie Agar, Keith L. Ligon, Mehdi Touat, Sandro Santagata. Phenogenomic characterization of immunomodulatory purinergic signaling in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1816.

High tumor mutational burden (hypermutation) is observed in some gliomas; however, its mechanisms of development and whether it predicts immunotherapy response are poorly understood. Here, we comprehensively analyze the molecular determinants of mutational burden and signatures in 10,294 gliomas including AACR Project GENIE and institutional datasets. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after temozolomide treatment. Experimentally, the mutational signature of post-treatment hypermutated gliomas was only recapitulated by temozolomide-induced damage in cells harboring MMR deficiency. MMR-deficient gliomas exhibited unique features including the lack of prominent T-cell infiltrates, extensive intratumoral heterogeneity, poor survival and low response rate to PD-1 blockade. Moreover, while microsatellite instability in MMR-deficient gliomas was not detected by bulk analyses, single-cell whole-genome sequencing of post-treatment hypermutated glioma cells demonstrated microsatellite mutations. This study shows that chemotherapy can drive acquisition of hypermutated populations without promoting response to PD-1 blockade and supports diagnostic use of mutational burden and signatures in cancer. Citation Format: Mehdi Touat, Yvonne Y. Li, Adam N. Boynton, Liam F. Spurr, Bryan Iorgulescu, Craig L. Bohrson, Isidro Cortes-Ciriano, Jack E. Geduldig, Kristine Pelton, Mary J. Lim-Fat, Sangita Pal, Shakti H. Ramkissoon, Frank Dubois, Charlotte Bellamy, Naomi Currimjee, Kenin Qian, Seth Malinowski, Aniket Shetty, Kin-Hoe Chow, Maïté Verreault, Erell Guillerm, Samy Ammari, Frédéric Beuvon, Karima Mokhtari, Agusti Alentorn, Caroline Dehais, Caroline Houillier, Florence Laigle-Donadey, Dimitri Psimaras, Alexandre Carpentier, Philippe Cornu, Laurent Capelle, Bertrand Mathon, Jill S. Barnholtz-Sloan, Arnab Chakravarti, Wenya L. Bi, Garrett M. Frampton, Marc Sanson, Brian M. Alexander, Andrew Cherniack, Patrick Y. Wen, David A. Reardon, Aurelien Marabelle, Peter J. Park, Ahmed Idbaih, Rameen Beroukhim, Pratiti Bandopadhayay, Franck Bielle, Keith L. Ligon. Mechanisms and therapeutic implications of hypermutation in gliomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5705.

Single-cell RNA sequencing (scRNAseq) has been performed across a range of intermediate to high-grade gliomas that each harbor multiple driver mutations. Pilocytic astrocytoma (PA), the most common childhood brain tumor, is a low-grade glioma. PAs frequently harbor oncogenic KIAA1549-BRAF fusions, and exhibit low rates of other driver mutations. While PAs exhibit a favorable prognosis compared to the higher-grade gliomas, treatment morbidity and tumor recurrence can represent major challenges for some PA patients. We performed scRNAseq of both tumor and non-tumor cells in newly-diagnosed PAs that contained KIAA1549-BRAF rearrangements. To confidently distinguish tumor cells from nontumor cells, we sorted cells by glial progenitor marker A2B5 status and profiled KIAA1549-BRAF fusion status of cells using a sensitive quantitative PCR-based assay. Results were validated using RNA in situ hybridization. When compared to higher-grade gliomas, a higher proportion of the PA tumor cells exhibited a differentiated, astrocyte-like phenotype. A smaller proportion of cells exhibited a progenitor-like phenotype with evidence of proliferation. These progenitor-like tumor cells expressed a mitogen-activated protein kinase (MAPK) program that was absent from higher-grade gliomas. Similar patterns of expression of genes associated with the astrocyte-like and MAPK gene programs were also seen in formalin fixed, paraffin embedded PA tissues using RNA in situ hybridization. Immune cells, especially microglia, comprised almost half of all cells in the PAs and accounted for differences in bulk expression profiles between tumor locations and subtypes. These single cell transcriptional data reveal a transcriptional developmental hierarchy in a pediatric low grade glioma that is skewed towards more mature brain cells compared to higher-grade gliomas. The results indicate that future analyses of bulk PA tissues should attempt to account for considerable infiltration by nontumor cells. Finally, the finding that a MAPK gene program is not uniformly expressed in PA tumor cells has implications for ongoing clinical investigations of therapies directed at the MAPK pathway for the treatment of PA. Citation Format: Zachary J. Reitman, Brenton Paolella, Guillaume Bergthold, Kristine Pelton, Robert Jones, Sarah Becker, Claire E. Sinai, Haley Malkin, Ying Huang, Leslie Grimmett, Zachary T. Herbert, Yu Sun, Jessica Weatherbee, John Alberta, John Daley, Orit Rozenblatt-Rosen, Rosalind Segal, Daphne Haas-Kogan, Mariella G. Filbin, Mario L. Suva, Aviv Regev, Charles Stiles, Mark W. Kieran, Liliana Goumnerova, Keith L. Ligon, Alex K. Shalek, Pratiti Bandopadhayay, Rameen Beroukhim. Single cell RNA sequencing reveals mitogenic and progenitor gene programs inBRAF-rearranged pilocytic astrocytomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3647.

Many children with metastatic or recurrent pediatric solid tumors continue to have poor survival, and there is an immense need to identify novel therapeutic approaches. Moreover, these cancers typically have simple genomes with limited known druggable molecular events. In order to discover new vulnerabilities in pediatric solid tumors, we have performed genome-scale CRISPR-Cas9 loss-of-function screening and deep “omic” characterization in over 60 pediatric cancer cell lines to date, including neuroblastoma, medulloblastoma, Ewing sarcoma, malignant rhabdoid tumor and rhabdomyosarcoma lines, to begin defining a pediatric cancer dependency map. Global analyses of the pediatric dependency landscape have identified emerging classes of pediatric cancers, including epigenetic-driven, aberrant transcription factor-driven and receptor tyrosine kinase-driven malignancies. For example, the preferential dependencies identified in a subset of neuroblastoma, which has aberrantly high expression of the transcription factor MYCN, are highly enriched for an interconnected network of genes annotated to have transcription factor activity. In addition to the global evaluation, we have developed methods and tools for prioritizing targets for further validation within a cancer type. These tools computationally integrate the pediatric dependency data across multiple datasets to identify categories of genetic dependencies that are especially strong hits or enriched hits in a specific pediatric malignancy. As an example, the intersection of MYCN-amplified neuroblastoma specific dependencies and H3-lysine 27 acetylation (H3K27ac) profiling across MYCN-amplified neuroblastoma allowed us to identify a transcriptional core regulatory circuit (CRC) that may drive the malignant state. Furthermore, targeting transcription with the BRD4 inhibitor JQ1 and CDK7 inhibitor THZ1 caused synergistic killing of neuroblastoma cells suggesting a novel therapeutic approach to treating this disease. Thus, defining a comprehensive pediatric cancer dependency map and developing the methods and tools to prioritize vulnerabilities in different cancer types will allow us to discover both novel biology and new therapeutic opportunities in childhood malignancies. Citation Format: Neekesh V. Dharia, Clare Malone, Amanda Balboni Iniguez, Lillian Guenther, Liying Chen, Gabriela Alexe, Adam D. Durbin, Mark W. Zimmerman, Andrew Hong, Pratiti Bandopadhayay, Mariella G. Filbin, Thomas Howard, Brenton Paolella, Iris Fung, Josephine Lee, Phil Montgomery, John M. Krill-Burger, Brian J. Abraham, Jennifer Roth, David E. Root, Richard A. Young, A. Thomas Look, Rameen Beroukhim, Jesse S. Boehm, William C. Hahn, Todd R. Golub, Aviad Tsherniak, Francisca Vazquez, Kimberly Stegmaier. Defining a pediatric cancer dependency map [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2352.

Diffuse pediatric low-grade gliomas (PLGGs) are among the most common solid tumors in children. While BRAF mutations and MYBL1 rearrangements have recently been identified as diagnostic and treatment related oncogenic drivers in pediatric gangliogliomas and diffuse astrocytomas, respectively, for the majority of diffuse PLGGs the oncogenic driver(s) remain unknown. Recent efforts to identify new oncogenic drivers lack sufficient power to determine the true frequency of alterations in genes or to associate specific alterations with rare histological subtypes, such as Angiocentric gliomas (AGs). To address this issue, we performed genomic analysis of new and published data from 249 PLGGs including 19 AGs. We identified MYB-QKI fusions as a specific, recurrent, single candidate driver event in AGs. Although MYB is expressed during early brain development in a subset of progenitor cells, it is not known to play a role in normal cortical brain where AGs frequently occur. In vitro functional studies show MYB-QKI rearrangements promote tumorigenesis in mouse neural stem cells (mNSCs) through three mechanisms: MYB activation by truncation, enhancer translocation driving aberrant MYB-QKI expression, and hemizygous loss of the tumor suppressor QKI. Expression of the MYB-QKI fusion increases mNSC proliferation and activates known MYB target genes including KIT and CDK6. H3K27ac enhancer profiling of AG tumors demonstrate active enhancer elements are translocated proximally to the MYB promoter while in vitro promoter assays confirmed that QKI enhancer sequences activate the MYB promoter. Together with functional studies demonstrating that MYB-QKI can also bind and activate the MYB promoter, these data support a positive auto-regulatory feed-back loop model in which active MYB-QKI is able to drive its own expression through enhanced MYB-promoter-activation. Furthermore, when injected orthotopically into immunodeficient mice, expression of truncated MYB or MYB-QKI is sufficient to induce tumor formation that recapitulate histologic and immunophenotypic characteristics of AGs. Analysis of AG specific MYB-QKI fusion represents the first example of how a single driver rearrangement simultaneously transforms cells via three genetic and epigenetic mechanisms in a cancer. Citation Format: Lori Ramkissoon, Pratiti Bandopadhayay, Payal Jain, Guillaume Bergthold, Adam Resnick, Rameen Beroukhim, Keith Ligon. MYB-QKI rearrangements in angiocentric glioma drive tumorigenicity through a tripartite mechanism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4372.

Genomic instability is a hallmark of cancer resulting in widespread somatic copy number alterations. We integrated a genome-scale shRNA viability screen and copy number profiles from 179 cancer cell lines to perform an unbiased analysis of copy-number associated gene-dependency interactions. We found most copy-number associated gene dependencies result from losses of genetic material rather than gains. Strikingly, the most enriched class of these dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes. Hemizygous loss of CYCLOPS genes sensitizes cancer cells to their further suppression. One of the “top hits” from the analysis was the pre-mRNA splicing factor SF3B1, which is also frequently mutated in cancer. We then sought to evaluate SF3B1 as a CYCLOPS gene. Cancer cells with hemizygous SF3B1 copy-loss were uniquely sensitive to partial SF3B1 suppression by RNAi compared to cells with normal SF3B1 gene dosage. Mechanistically, cancer cells harboring partial SF3B1 copy-loss lack a reservoir of excess SF3b complex, a precursor complex of the U2 snRNP, which protects cells with normal SF3B1 copy number from cell death upon SF3B1 suppression. Our data highlight the prevalence of CYCLOPS dependencies in cancer and establish the spliceosome as a frequent CYCLOPS target. Further, these data indicate targeting wild-type SF3B1 as a novel cancer dependency in cells with hemizygous SF3B1 copy-loss. Citation Format: Brenton R. Paolella, William J. Gibson, Laura M. Urbanski, John A. Alberta, Travis I. Zack, Pratiti Bandopadhayay, Caitlin A. Nichols, Pankaj K. Agarwalla, Meredith S. Brown, Rebecca Lamothe, Yong Yu, Peter S. Choi, Esther A. Obeng, Dirk Heckl, Benjamin L. Ebert, Guo Wei, Belinda Wang, William C. Hahn, Francisca Vazquez, Barbara A. Weir, Charles D. Stiles, Robin Reed, Rameen Beroukhim. Genome-wide copy number dependency analysis identifies partial copy loss of SF3B1 as a novel cancer vulnerability. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4369.