bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A77: Targeting lymphocyte exclusion in metastatic osteosarcoma
Authors: Sorenson Laurie , McEachron Troy A.
Published: August 2020

Osteosarcoma (OS) is the most common bone tumor in pediatric and adolescent/young adult patients. Over the past three decades, significant improvements in the survival rates or therapeutic approaches for these patients have not been made, especially in the context of metastatic disease. While immune checkpoint blockade has revolutionized the therapeutic landscape in various adult malignancies, its impact in OS has been largely underwhelming. Currently, it is unknown whether the lack of therapeutic benefit of immune checkpoint inhibition observed in patients with OS is truly due to treatment inefficacy rather than a limited understanding of the tumor microenvironment that supports this aggressive disease. To address this knowledge gap, we have performed targeted gene expression profiling of metastatic and nonmetastatic osteosarcoma specimens. Our data demonstrate that T cells are largely excluded from the metastatic specimens and that this exclusion significantly correlates with markers of vascular instability. In a pathologic setting, such as that of cancer, VEGF and ANG2 signaling promote vascular instability, which limits leukocyte extravasation and subsequent tumor infiltration. Our data suggest that vascular destabilization mediated by VEGF/ANG2 signaling impedes T-cell infiltration specifically in metastatic OS and identify these molecules as potential targets for therapeutic intervention.

Citation Format: Laurie Sorenson, Troy A. McEachron. Targeting lymphocyte exclusion in metastatic osteosarcoma [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 A77.
Abstract B08: Beyond synthetic lethality: Multiple mechanisms can explain genetic interactions within childhood cancer
Authors: Daub Josephine T. , Amini Saman , Holstege Frank C.P. , Kemmeren Patrick
Published: August 2020

Even though the survival rate of childhood cancer has increased in the last decades to around 80% today, it is still a leading cause of death for children in developed countries. Cancer develops through the acquisition of multiple mutations, and it is assumed that genetic interactions between mutated genes play an important role in cancer onset and progression. One approach to find genetic interactions in cancer is to search for pairs of mutated genes that occur more (or less) often than expected given the frequency of the individual mutated genes. Highly co-occurring mutated genes suggest a cooperative role of these altered genes in cancer development. Mutually exclusive gene pairs can be a signal of synthetic lethality, where the combination of the two mutations is lethal for the cancer cells, and could therefore point to possible cancer treatments. We developed a statistical pipeline, based on two genetic interaction tests, to detect significant cases of co-occurrence and mutual exclusivity in two pediatric cancer data sets, comprising over 2,500 tumors from 23 cancer types. In total we detect 15 co-occurring and 26 mutually exclusive candidate genetic interactions. Nearly all candidate gene pairs are uniquely found in one cancer type, supporting earlier findings that genetic interactions are often cell or cancer type specific. Interestingly, we find that synthetic lethality is not the main cause of the mutual exclusivity patterns in our data set. The majority of these patterns can be attributed to cancer subtypes and within pathway relationships, where the effect of a mutated gene is similar to mutating a (downstream) gene in the same pathway. This initial map of genetic interactions in childhood cancer provides a solid starting point to select suitable candidates for experimental validation as well as extending the analyses to investigate the contribution of genetic interactions towards structural variants, genetic predisposition, and cellular pathways.

Citation Format: Josephine T. Daub, Saman Amini, Frank C.P. Holstege, Patrick Kemmeren. Beyond synthetic lethality: Multiple mechanisms can explain genetic interactions within childhood cancer [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 B08.
Abstract B09: Epigenetic changes mediated by H3.3 G34R mutation in a CRISPR-edited pediatric glioblastoma cell line
Authors: Deshmukh Shriya , Chen Carol C.L. , Ouanouki Amira , Jabado Nada
Published: August 2020

Background: Pediatric glioblastomas (pGBM) are aggressive and lethal brain tumors with few effective treatment options. Since the discovery of recurrent histone H3 mutations in pGBMs, multiple groups have sought to uncover the mechanism by which these mutations drive tumorigenesis. One striking aspect of histone H3 mutations is their tumor location and age-specificity: the K27M mutation occurs in midline brain structures in infants and young children, whereas the G34R/V mutations occur in the cerebral hemisphere of adolescents. Efforts to elucidate the mechanism underlying K27M tumorigenesis have centered on K27M-mediated inhibition of the PRC2 complex and the resulting drastic loss of the repressive H3K27me3 mark in these tumors. Studies have shown that G34 mutations affect methylation of the adjacent K36 residue, resulting in a local decrease of the active H3K36me3 mark on the mutant histone. It remains uncertain whether the G34R/V mutations promote global changes in the epigenome that drive tumorigenesis.

Methods: To investigate the epigenetic consequences of the G34R mutation, we generated an isogenic in vitro model using CRISPR/Cas9 to correct the G34R mutation in a pGBM-derived cell line. We then comprehensively characterized and compared the epigenome and transcriptome of CRISPR-unedited clones (carrying the G34R mutation) and CRISPR-edited (wild-type) clones. Using ChIP-seq, we profiled H3K36me3 and H3K27me3 marks to study G34R-mediated regulation of these antagonistic marks.

Results: ChIP-seq profiling revealed that CRISPR editing of the G34R mutation had minimal effects on total levels or genomic distribution and profile of H3K36me3 and H3K27me3 marks. In contrast, we previously showed that CRISPR editing of the K27M mutation in pGBM cell lines rescued total H3K27me3 levels and genomic distribution to that of histone wild-type pGBMs. In agreement with their similar epigenetic profiles, there were very few transcriptomic differences between G34R and wildtype cells.

Conclusions: Our results suggest that the epigenome in G34R-mutant pGBM cells may be less dynamic or amenable to epigenetic perturbation from removal of the initial driver oncomutation than K27M-mutant tumors. These findings underscore that different strategies may be required to target and treat G34R and K27M-mutant pGBM tumors.

Citation Format: Shriya Deshmukh, Carol C.L. Chen, Amira Ouanouki, Nada Jabado. Epigenetic changes mediated by H3.3 G34R mutation in a CRISPR-edited pediatric glioblastoma cell line [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 B09.
Abstract B17: A C19MC-LIN28A-MYCN oncogenic circuit driven by hijacked super-enhancers is a distinct therapeutic vulnerability in ETMRs—a lethal brain tumor
Authors: Mumal Iqra , Sin-Chan Patrick , Suwal Tannu , Singh Irtisha , Li Xiao-Nan , Lin Charles , Mack Stephen , Rich Jeremy , Huang Annie
Published: August 2020

Embryonal tumors with multilayered rosettes (ETMRs) are distinctly challenging brain tumors of infants and very young children, with characteristic rapid progression and only 10-20% overall survival. ETMRs have characteristic amplification of Chr19q13.41 miRNA cluster (C19MC) and enrichment of pluripotency factor LIN28A. Since the discovery of C19MC, an embryonic stem cell-enriched, primate specific miRNA cluster, as a disease marker of ETMR, there has been limited progress in biologic and therapeutic understanding of this disease. In prior studies we demonstrated single C19MC oncomiRs promotes oncogenesis and inhibit neural differentiation, yet the specific mechanisms of C19MC remains unclear. Here we show expression of 5-C19MC oncomiRs cooperatively inhibits multiple cell cycle checkpoint tumor suppressors to drive cellular proliferation. We further show that C19MC oncomiRs activate a potent oncogenic circuit by upregulating MYCN and LIN28A via downregulation of RNA binding protein, Tristetraprolin. Using RNA-IP analyses, we uncovered epigenetic regulators as major targets of LIN28A and showed that LIN28A directly regulates expression of embryonic-neural DNMT3A2 and DNMT3B6 isoforms. Deep epigenetic mapping on primary tumors revealed high DNA-DNA interactions and enhancer hijacking focused on MYCN. We also discovered tumors with TTYH1-C19MC gene fusions harbored a novel hybrid super-enhancer. Lastly, super-enhancer analysis revealed MYCN/MAZ as major transcriptional regulators and that treatment with a potent MYCN and bromodomain inhibitor JQ-1 inhibited proliferation of primary cells. Collectively, our data reveal that tumor-specific genomic and epigenomic alterations of C19MC entrap and drive multiple feed-forward loops to fuel a potent C19MC-LIN28A-MYCN oncogenic circuit, that can be powerfully abrogated by bromodomain inhibitors. Our findings underscore C19MC as a critical oncogene in ETMRs and provide critical therapeutic insights and a framework for developing high-fidelity models for this orphan disease.

Citation Format: Iqra Mumal, Patrick Sin-Chan, Tannu Suwal, Irtisha Singh, Xiao-Nan Li, Charles Lin, Stephen Mack, Jeremy Rich, Annie Huang. A C19MC-LIN28A-MYCN oncogenic circuit driven by hijacked super-enhancers is a distinct therapeutic vulnerability in ETMRs—a lethal brain tumor [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 B17.
Abstract B19: Identification of physiologically relevant EWS-FLI1 target genes in Ewing’s sarcoma via CRISPRa screening
Authors: Saratov Vadim , Ngo Qui A. , Pedot Gloria , Niggli Felix K. , Schaefer Beat W.
Published: August 2020

Ewing’s sarcoma is an aggressive pediatric bone and soft-tissue cancer with a pathognomonic chromosomal translocation t(11;22) resulting in expression of EWS-FLI1, an “undruggable” fusion protein acting as a transcriptional modulator. Identification and ranking of repressed EWS-FLI1 target genes essential for cancer cell survival will potentially provide much-needed insights to develop novel therapeutic strategies. We performed a CRISPR activation (CRISPRa) dropout screen in Ewing cells. We generated a clonal SKNMC cell line homogenously expressing the synergistic activation mediator (SAM) CRISPRa system to functionally interrogate repressed EWS-FLI1 target genes. The systems functionality was tested using CD44, a surface marker absent on the surface of Ewing cells. We found robust and stable expression of CD44 after introduction of promoter-targeting gRNAs. The library of repressed EWS-FLI1 target genes, named LIBerty, was constructed to target 872 genes bioinformatically selected from publicly available silenced EWS-FLI1 RNA-Seq datasets as well as genes identified as repressed signature genes in Ewing’s sarcoma via meta-analysis with 3,777 unique guideRNAs. LIBerty was lentivirally delivered into SAM SKNMC cells and data from four biologic replicates were gathered at three time points: three, ten, and 21 days after infection. Cells were harvested and samples were prepared for next-generation sequencing (NGS) via PCR. The NGS data were evaluated with the analysis tool PinAPL-Py. Preliminary analysis of the screen revealed efficient selection of the positive control genes BAD and BBC3. In addition, high-ranking hits included CDKN1A and CDKN1C, both cell cycle regulators and known tumor-suppressor genes. Furthermore, we identified PCDH7 and TGFBR2, two genes already known from the literature to play a role in Ewing’s sarcoma. The presence of validated genes amid highest-ranking candidates confirms robustness of the conducted CRISPRa screen. Further analysis and validation of previously unexplored targets and pathways is currently ongoing. Our CRISPRa screen revealed both known as well as previously unknown EWS-FLI1 repressed genes whose absence of function is essential for tumor cell survival.

Citation Format: Vadim Saratov, Qui A. Ngo, Gloria Pedot, Felix K. Niggli, Beat W. Schaefer. Identification of physiologically relevant EWS-FLI1 target genes in Ewing’s sarcoma via CRISPRa screening [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 B19.
Abstract B34: Cord blood CD34+ HSPCs: An in vitro model system for characterizing NUP98 fusions
Authors: Hiltenbrand Ryan , Miller Jonathan , Klco Jeffery
Published: August 2020

Chromosomal translocations of the Nucleoporin 98 gene (NUP98) are associated with multiple hematopoietic malignancies. In particular, NUP98 rearrangements can be found in up to 6.6% of de novo cases of pediatric AML. Specific subpopulations of pediatric AML appear to be even more enriched for NUP98 fusions, with over 16% of cytogenetically normal pediatric AML patients and 15% of non-Down syndrome acute megakaryoblastic leukemia (AMKL) patients harboring these fusions. Clinically, the NUP98-KDM5A fusion is associated with erythroid and megakaryocytic leukemias while the NUP98-NSD1 fusion is associated with myelomonocytic leukemias. When NUP98 fusions are present in AML, they confer a poor prognosis. To better characterize the role of these fusions in human hematopoiesis, we used cord blood CD34+ (cb-CD34+) hematopoietic stem and progenitor cells (HSPCs) as an in vitro model system. We transduced cb-CD34+ HSPCs with NUP98-NSD1, NUP98-KDM5A, or empty vector control and studied the effects on cell proliferation and differentiation in liquid culture media containing cytokines (IL-6, FLT3-Ligand, SCF, TPO, and SR1 (an aryl hydrocarbon receptor antagonist)). NUP98-NSD1 and NUP98-KDM5A fusions increase the proliferation rate in liquid culture by three-fold and 19-fold, respectively (p<0.05). Consistent with observations from patients with these fusions, cells expressing NUP98-KDM5A have increased expression of CD235A and CD71, consistent with erythroid differentiation (p<0.05). In addition, cells expressing NUP98-NSD1 have increased expression of CD11b compared to NUP98-KDM5A (p<0.01) expressing cells. When placed in methylcellulose, the NUP98 fusions tested had no effect on total colony number, but we observed more erythroid differentiation for NUP98-KDM5A compared to vector control (p<0.05). Overall, the results of these in vitro studies demonstrate increased proliferation and inhibition of differentiation in HSPCs by NUP98 fusions, and cell differentiation patterns parallel the different AML subtypes observed in patients with NUP98-NSD1 and NUP98-KDM5A fusions. We propose cbCD34+ HSPCs as a promising system for in vitro modeling of NUP98 fusions.

Citation Format: Ryan Hiltenbrand, Jonathan Miller, Jeffery Klco. Cord blood CD34+ HSPCs: An in vitro model system for characterizing NUP98 fusions [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 B34.
Abstract B36: A novel model of osteosarcomagenesis reveals dysregulation of oxidative phosphorylation
Authors: Jewell Brittany E. , Xu An , Zhou Ruoji , Zhu Dandan , Lu Linchao , Zhao Ruying , Wang Lisa L. , Lee Dung-Fang
Published: August 2020

The purpose of this study is to develop a new model to study osteosarcoma in order to elucidate cellular pathways important in osteosarcomagenesis that are potential therapeutic targets. Osteosarcoma is the most common bone malignancy in childhood and early adolescence. Thirty percent of patients with the rare genetic disorder Type II Rothmund-Thomson syndrome (RTS) develop osteosarcoma. Patients with RTS have biallelic mutations in the RECQL4 gene, which encodes an ATP-dependent DNA helicase. Unfortunately, many attempts to model RTS-associated osteosarcoma have not been successful. Here, we describe a patient-derived induced pluripotent stem cell (iPSC) model capable of recapitulating the process of osteosarcomagenesis. Briefly, RTS patient fibroblasts are reprogrammed to iPSCs, then subsequently differentiated to mesenchymal stem cells. These cells are validated and further differentiated to early and late osteoblasts, the potential precursors of osteosarcoma. Because the iPSC-derived osteoblasts retain the genetic fidelity of the patient from which they were derived, we believe these cells are the ideal model to study the molecular mechanism associated with RTS-associated osteosarcoma. Utilizing the patient-derived osteoblasts, we have performed RNAseq and subsequent GSEA analyses to explore the transcriptome of RTS osteoblasts and found the dysregulation of oxidative phosphorylation in RTS osteoblasts. Interestingly, it has been reported that specific vacuolar ATPases are upregulated in patient cells. It has been reported that vacuolar ATPases function to increase proton concentration within the mitochondria and also within the extracellular environment. Understanding key mechanisms of osteosarcomagenesis is critical to making steps toward novel therapeutics.

Citation Format: Brittany E. Jewell, An Xu, Ruoji Zhou, Dandan Zhu, Linchao Lu, Ruying Zhao, Lisa L. Wang, Dung-Fang Lee. A novel model of osteosarcomagenesis reveals dysregulation of oxidative phosphorylation [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 B36.
Abstract B41: Genomic landscape of somatic mutations in osteosarcomas
Authors: Pires Sara F. , Costa Silvia S. , Vidal Daniel O. , Lengert André van Helvoort , Boldrini Érica , da Silva Sandra R.M. , Rosenberg Carla , Lopes Luiz F. , Maschietto Mariana , Krepischi Ana
Published: August 2020

Pediatric cancers are among the leading causes of childhood mortality, with osteosarcomas being the most prevalent bone tumors, characterized by an aggressive clinical course. However, knowledge about the molecular basis of osteosarcomas is still limited, hampering advances in diagnosis and treatment. In this context, we characterized the genomic landscape of somatic mutations in 28 osteosarcomas (24 pediatric patients and 4 adults) aiming to delineate the panel of prevalent mutations in Brazilian patients. Genomic libraries of these osteosarcoma samples were constructed using the TruSight One panel (Illumina), covering 4,813 clinically relevant genes, including known cancer genes. Generated data at high coverage (median >100x depth coverage) were annotated based on public databases containing population variant frequencies (including the Brazilian ABRAOM), as well as clinical information. For further analysis, we filtered only coding nonsynonymous variants absent from both population databases and an additional pool of 20 nonrelated germline samples. In total, 728 variants were identified mapping in 606 genes, with an average rate of 41 mutations per tumor. The panel of detected variants was composed by 93 loss-of-function mutations (38 frameshift, 25 splicing, and 30 premature stop codon) and 635 missense (43 inframe insertions/deletions and 592 single-nucleotide variations), some of them already reported in cancer databases (27 in COSMIC and 39 in ICGC). From 606 genes, 25 were previously associated with osteosarcomas, and they are related to apoptosis, cell growth and differentiation, DNA repair, transcriptional regulation, and tumor-suppression mechanisms. The most frequent mutations were detected in RB1 and TP53; other recurrently mutated genes were mostly linked to biologic pathways potentially related to the disease onset, such as hormonal response (AR), transcriptional regulation and muscle differentiation (FRG1), DNA repair (HERC2), cell signaling (KIR2DL4), and cell proliferation/differentiation (PTPRQ). Additionally, the tumors presented a highly complex pattern of copy number alterations, some of them resembling chromothripsis. This is the first assessment of a Brazilian cohort of osteosarcomas, and functional studies will be further applied to better understand the role of the disclosed mutations for tumor biology.

Citation Format: Sara F. Pires, Silvia S. Costa, Daniel O. Vidal, André van Helvoort Lengert, Érica Boldrini, Sandra R.M. da Silva, Carla Rosenberg, Luiz F. Lopes, Mariana Maschietto, Ana Krepischi. Genomic landscape of somatic mutations in osteosarcomas [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 B41.
Abstract B45: Therapeutic potential of splicing in RMS: SRSF2 binding modulation controls MDM2 alternative splicing
Authors: Montes Matias I. , Comiskey Daniel F. , Khurshid Safiya , Chandler Dawn S.
Published: August 2020

MDM2 undergoes complex alternative splicing and one of its spliced isoforms, MDM2-ALT1, comprised of coding exons 3 and 12, is highly expressed in pediatric rhabdomyosarcoma, and its expression correlates with a poor disease prognosis. MDM2-ALT1 expression is also upregulated under conditions of cellular genotoxic stress, and this alternative splicing is conserved in the mouse Mdm2 gene. Furthermore, previous research from our lab has reported that the splicing factors SRSF1, FUBP1, and PTBP1 are involved in this regulation; nevertheless, the way that MDM2 splicing is controlled is not entirely understood. In this work, we explore the role of the splicing factor SRSF2/SC35 in the alternative splicing of MDM2, hypothesizing that this factor acts as a positive regulator, in both human and mouse, facilitating the production of the full-length MDM2 mature transcript. This splicing factor has been reported to be mutated in several cancers, including myelodysplastic syndromes and rhabdomyosarcoma. Moreover, using a minigene system that mimics the endogenous Mdm2 splicing in response to UV and cisplatinum-induced DNA damage and the CRISPR-Cas9 technology, we show that mutations in SRSF2 predicted binding sites alter Mdm2 alternative splicing. The CRISPR-engineered mutant cells that express this splice isoform show increased proliferation in an Arf/p19 null context. On the other hand, we show that by using antisense oligonucleotides working as splice-switching molecules that target the SRSF2 binding sites in the MDM2 pre-mRNA, we are able to shift the expression of the MDM2 splice isoforms to drive p53 target gene expression in p53 wild-type cells. Furthermore, in an effort to understand better the effect of the MDM2-ALT1 isoform in carcinogenesis, we performed the CRISPR-Cas9 technique to generate a mouse model expressing the cancer-associated spliced isoform. We expect the mouse to be a cancer-sensitized model that will give rise to RMS tumors and provide a preclinical model for testing splice-switching oligonucleotide therapies.

Citation Format: Matias I. Montes, Daniel F. Comiskey, Safiya Khurshid, Dawn S. Chandler. Therapeutic potential of splicing in RMS: SRSF2 binding modulation controls MDM2 alternative splicing [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 B45.
Abstract B56: A CRISPR/Cas9 domain screen identifies a small motif in the PAX3-FOXO1 transactivation domain relevant for tumor maintenance in alveolar rhabdomyosarcoma
Authors: Wachtel Marco , Benischke Katharina , Schäfer Beat W.
Published: August 2020

Fusion transcription factors are the main drivers of tumorigenesis in many pediatric leukemias and sarcomas. One example is PAX3-FOXO1 (P3F), the pathognomonic marker of alveolar rhabdomyosarcoma (aRMS). Since these tumors are often also characterized by a quiet mutational landscape and a paucity of druggable oncogenes, the fusion proteins often remain as the only relevant drug target. However, transcription factors are challenging targets for small-molecule inhibitors, since they lack an enzymatic activity and, apart from the DNA binding domains, are usually intrinsically disordered. Identification of druggable structures in such proteins is therefore of great clinical interest. Our goal of this study was to identify new elements important for P3F activity. Towards this aim, we made use of the CRISPR/Cas9 technology and performed a saturated screen for identification of important functional domains in P3F in the endogenous context. This revealed that both DNA binding domains are indispensable, as expected for a transcription factor. Importantly, however, the screen also identified a 40-amino-acid-long domain at the C-terminus of the FOXO1 part to be strictly required for the function of the protein. Using a series of mutations in this domain in reporter assays, we identified a single cysteine whose mutation leads to more than 50% reduction in transcriptional activity. In addition, cells efficiently differentiated and stopped proliferating when this Cys was mutated in aRMS cells. These results suggest that functional interference with this domain could be a promising approach for aRMS therapy.

Citation Format: Marco Wachtel, Katharina Benischke, Beat W. Schäfer. A CRISPR/Cas9 domain screen identifies a small motif in the PAX3-FOXO1 transactivation domain relevant for tumor maintenance in alveolar rhabdomyosarcoma [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 B56.
Abstract B57: A combination of IL7Rα and NRAS mutations sheds light on the oncogenesis of T-cell acute lymphoblastic leukemia in a murine model
Authors: Winer Hila , Rodrigues Gisele , Li Wenqing , Hixon Julie , Durum Scott
Published: August 2020

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. While most cases of ALL arise from B-cell precursors, 15% originate from T cells in the thymus (T-ALL). With current treatments, survival rate is high, but relapse occurs in 15-20% of children. In addition, most treatments are highly toxic, with severe side effects. Approximately 10% of T-ALL patients have somatic gain-of-function mutations in IL7Rα gene, and 15-22% of ALL patients have mutations in NRAS or KRAS. Furthermore, Ras pathway mutations are responsible for 30%-50% of ALL relapse. Recently, our lab showed that mutations in human IL7Rα alone (c.731_732insTTGTCCCAC) or human NRAS alone (G13D) are not sufficient to induce T-ALL from primary cells. However, a combination of both causes severe T-ALL in mice. Treatment with inhibitors against IL7Rα and NRAS signaling led to prolonged survival in these mice. Our new data indicate that the correct ratio between IL7Rα and NRAS expression is necessary to generate T-ALL in mice. Using RAG-/- mice injected with primary thymocytes transduced with mutated IL7Rα and NRAS in the optimal sequence in a polycistronic vector led to fatal disease 3 weeks after injection. These mice showed a 30-fold increase of leukocytes in the blood, spleen, and thymus, 80-fold increase in the lymph nodes, and a 5-fold increase in spleen weight, all indications of severe T-ALL. There was no indication of disease progression in mice injected with either empty vector or wild-type IL7Rα and mutated NRAS. To identify the pathways involved in T-ALL by these mutations, we used mass spectrometry and RNA sequencing. Our analysis showed significant increases in the well-known antiapoptotic, B-cell lymphoma 2 (BCL-2), mRNA, and protein levels and a major decrease in the proapoptotic BCL-2 antagonist/killer 1 (BAK-1) protein levels. In this manner, combination of both IL7Rα and NRAS mutations resulted in high Bcl-2:Bak-1 ratio, supported also by Western blot analysis. In addition, we observed a significant increase in the protein cytokinesis midbody regulator citron kinase (Cit), potentially leading to increased thymocytes proliferation and driving T-ALL formation. Taken together, our lab developed a strong model to study T-ALL in mice, shedding light on novel pathways of oncogenesis and targets for better therapeutic development.

Citation Format: Hila Winer, Gisele Rodrigues, Wenqing Li, Julie Hixon, Scott Durum. A combination of IL7Rα and NRAS mutations sheds light on the oncogenesis of T-cell acute lymphoblastic leukemia in a murine model [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 B57.
Abstract IA04: New approaches to study cancer predisposition syndromes
Authors: Kratz Christian P.
Published: August 2020

The diagnosis of a cancer predisposition syndrome (CPS) in a child diagnosed with cancer influences medical care; however, many open questions remain in the areas of counseling, psychosocial support, cancer prevention, surveillance, and therapy. To address such issues, a CPS-registry was launched: www.cancer-predisposition.org. The CPS-registry (1) provides detailed information for affected families and health care professionals, (2) collects biospecimens for translational research, (3) assembles both retrospective and annual prospective data on cancer diagnoses and surveillance measures in individuals with approximately 60 different CPS, (4) makes mutation data publicly available through accessible databases, (5) shares data internationally, (6) links data with trial groups and cancer registries, and (7) provides expert opinion for individual treatment decisions. Due to hidden clinical signs, CPS are often overlooked; however, various clinical tools have been developed to increase the percentage of families that are being offered counseling and testing. Moreover, CPS diagnosis is dramatically facilitated by the increasing use of agnostic next-generation sequencing in pediatric oncology, supporting the notion that 10% of children with cancer harbor mutations in cancer predisposition genes, often in the absence of clinical signs. CPS diagnosis yet remains a challenge prior to a first cancer diagnosis; however, a more systematic diagnostic approach is required to offer prevention and surveillance measures to these individuals. Strong family support organizations such as LFSA and FARF have dramatically improved collaborative research in the CPS field. In the future, clinical protocols for CPS individuals will pave the way for better prevention, surveillance, and cancer treatment strategies.

Citation Format: Christian P. Kratz. New approaches to study cancer predisposition syndromes [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 IA04.
Abstract IA05: Circulating tumor DNA as a tool for prognostication, translational discovery, and early cancer detection
Authors: Crompton Brian
Published: August 2020

New sequencing technologies have facilitated the detection and profiling of circulating tumor DNA (ctDNA) in the blood of patients with cancer. Recent efforts have demonstrated that ctDNA can be detected in the blood of pediatric patients with solid malignancies, using assays specifically designed for the unique somatic profiles observed in these cancers. Once identified in a blood sample, ctDNA levels appear to correlate with outcome and can be tracked over time to measure response to therapy and detect recurrence. Deep profiling of ctDNA can identify subclonal somatic variants that are not detectable in single-tumor biopsy samples, enabling a broader understanding of tumor heterogeneity and the ability to discover novel patterns of tumor evolution and treatment resistance. Validation of the prognostic and translational value of these assays requires leveraging large prospective studies, and this work is ongoing. One obvious application of this new technology is the potential to detect the presence of cancer in pediatric patients with increased cancer risks, including cancer predisposition syndromes. Collaborative multi-institutional efforts are under way to collect samples and develop new ctDNA assays for early cancer detection.

Citation Format: Brian Crompton. Circulating tumor DNA as a tool for prognostication, translational discovery, and early cancer detection [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 IA05.
Abstract IA09: Immunotherapeutic approaches for pediatric solid tumors
Authors: Handgretinger Rupert
Published: August 2020

For patients with primary metastatic or recurrent soft tissue sarcomas, cytotoxic chemotherapy is currently the primary treatment modality. Despite intensive chemotherapy, the outcome is poor and additional new therapeutic strategies in combination with chemotherapy have to be investigated. Immunotherapy, which involves the patient’s own or a new allogeneic immune system, might have great potential to improve the outcome of affected patients. We have focused on the use of haploidentical transplantation as a form of intensive immunotherapy in order to utilize a graft-versus-solid tumor effect, which might be exerted by donor-derived alloreactive Natural Killer (NK) cells, but also by donor-derived T cells. In a first pilot trial, we have transplanted 26 patients with relapsed neuroblastoma from a haploidentical donor. The transplant-related mortality (TRM) was 0% and the overall survival (OS) was 20%. We then started a protocol using haploidentical transplantation in 68 patients followed post-transplant by 6-9 cycles of the monoclonal anti-GD2 antibody ch14.18CHO. The TRM was 5% and the 3-year OS is 58%. In addition to neuroblastoma, we also hope to apply similar strategies to other pediatric advanced solid tumors including Ewing’s sarcoma, rhabdomyosarcoma, and others. We conclude that haploidentical transplantation of patients with relapsed/refractory solid tumors is a platform on which further immunotherapeutic strategies can be developed, such as antibodies directed against other targets than GD2, such as CD276, or bispecific T-cell engagers (BiTEs), chimeric antigen receptor (CAR) T-cells or CAR NK cells, or antibody/cytokine fusion proteins, and these approaches will be discussed. The main goal of immunotherapy, however, must be to establish a long-term memory immune response against the tumor, and haploidentical transplantation might facilitate such a response.

Citation Format: Rupert Handgretinger. Immunotherapeutic approaches for pediatric solid 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 IA09.
Abstract IA17: From functional genomics to new targets in pediatric oncology
Authors: Stegmaier Kimberly
Published: August 2020

The sequencing of human genomes harkened a new era of pediatric cancer investigations. Indeed, genomic discoveries, such as the identification of ALK mutations in neuroblastoma, Philadelphia-like fusions in acute B-cell acute lymphoblastic leukemia (B-ALL), and NTRK fusions in infantile fibrosarcoma, have led to early successes in the application of genomically informed, targeted therapy for children with cancer. While these new drugs have become an important part of the pediatric cancer armamentarium, many children with cancer still lack targeted therapies. Thus, new therapeutic approaches are still needed. Approaches such as RNA interference and CRISPR-Cas9 have transformed the functionalizing of the genome and offer promise for identifying new cancer therapeutic targets. We have applied genome-scale CRISPR-CAS9 screening to identify novel therapeutic targets in pediatric malignancies through the development of a Pediatric Cancer Dependency Map. We screened the Broad Institute’s Avana library in over 100 pediatric cancer cell lines and compared their dependencies to over 600 nonpediatric cell lines. Novel targets emerging from these efforts will be discussed.

Citation Format: Kimberly Stegmaier. From functional genomics to new targets in pediatric oncology [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 IA17.
Abstract IA18: Clinically relevant mutational signatures in childhood cancer
Authors: Shlien Adam
Published: August 2020

Mutagenic processes leave imprints on the genome in the form of mutations in a specific nucleotide context, forming a unique signature. The driving forces of rarer signatures, especially those seen in relapsed childhood cancers, are mostly unknown. In this talk, I will present data showing the contribution of intrinsic and extrinsic mutators to childhood cancer, and analyses using mutation signatures to accurately predict past mutagen exposure and inform therapeutic choice for ongoing patients.

Citation Format: Adam Shlien. Clinically relevant mutational signatures in childhood cancer [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 IA18.
Abstract IA27: Patient-derived organoids in pediatric cancer research
Authors: Calandrini Camilla , Schutgens Frans , Oka Rurika , Margaritis Thanasis , Candelli Tito , Mathijsen Luka , Ammerlaan Carola , van Ineveld Ravian , Derakhshan Sepideh , Custers Lars , Lijnzaad Philip , Begthel Harry , Kerstens Hinri , Rookmaker Maarten , Verhaar Marianne , Kemmeren Patrick , de Krijger Ronald , Pritchard-Jones Kathy , Rios Anne , van den Heuvel-Eibrink Marry , Holstege Frank , van Boxtel Ruben , Clevers Hans , Drost Jarno
Published: August 2020

Recent advances in in vitro culture technologies, such as adult stem cell-derived organoids, have opened up new avenues for the development of novel, more physiologic human cancer models. Such preclinical models are essential for efficient translation of basic cancer research into novel treatment regimens. We succeeded in growing organoids from a range of pediatric solid tumors, including Wilms’ tumors, renal cell carcinomas, and different types of rhabdoid tumors (i.e., AT/RT, MRT). Tumor organoids retain many characteristics of parental tumor tissue. For instance, Wilms’ tumor organoids retain the cellular heterogeneity of tumors, as they are composed of an intricate network of different cell types. Moreover, we demonstrate that tumor organoids are amenable to gene editing and high-throughput drug screens. In conclusion, our pediatric cancer organoids capture disease and tissue heterogeneity and provide a platform for basic cancer research, drug screening, and personalized medicine.

Citation Format: Camilla Calandrini, Frans Schutgens, Rurika Oka, Thanasis Margaritis, Tito Candelli, Luka Mathijsen, Carola Ammerlaan, Ravian van Ineveld, Sepideh Derakhshan, Lars Custers, Philip Lijnzaad, Harry Begthel, Hinri Kerstens, Maarten Rookmaker, Marianne Verhaar, Patrick Kemmeren, Ronald de Krijger, Kathy Pritchard-Jones, Anne Rios, Marry van den Heuvel-Eibrink, Frank Holstege, Ruben van Boxtel, Hans Clevers, Jarno Drost. Patient-derived organoids in pediatric cancer research [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 IA27.
Abstract IA29: Developmental and oncogenic programs in pediatric brain tumors dissected by single-cell RNA-sequencing
Authors: Filbin Mariella G.
Published: August 2020

During development, the multiple cell types of human brain arise from pluripotent stem cells via progressively more committed progenitor cells. Emerging data suggest that pediatric brain tumors arise from progenitors that have become developmentally stalled. In preliminary studies, our lab used single-cell RNA-seq to display the expression profiles of individual cells within pediatric midline and hemispheric high-grade gliomas. Our data show that these tumors are composed of (i) replicating cells that resemble glial and neural progenitors and (ii) maturing cells resembling astrocytes, oligodendrocytes, or mesenchymal cells. In this presentation I will address how single-cell RNA-sequencing has revolutionized our understanding of the developmental hierarchy and oncogenic programs in pediatric brain tumors. Our recent findings pointing towards potential cells of origin of midline and hemispheric pediatric high-grade gliomas as well as plasticity of different cancer cell states will be discussed.

Citation Format: Mariella G. Filbin. Developmental and oncogenic programs in pediatric brain tumors dissected by single-cell RNA-sequencing [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 IA29.
Abstract PR09: STAG2 mutations alter topologic organization of the genome and cis-mediated interactions
Authors: Surdez Didier , Zaidi Sakina , Grossetête Sandrine , Raynal Virginie , Baulande Sylvain , Hill Véronique , Delattre Olivier
Published: August 2020

STAG2 is one of the most recurrently mutated genes in human cancer, including in Ewing sarcoma, an aggressive bone tumor driven by the chimeric EWSR1-FLI1 transcription factor. We and others have shown that STAG2 mutations occurs in approximately 20% of Ewing sarcoma, leading most frequently to complete loss of function (LOF) of its protein. We also showed that STAG2 mutations were associated with poor outcome for these patients. However, functional mechanisms associated with these mutations are still poorly understood. STAG2 encodes an integral member of the cohesin complex, a ring-shaped multiprotein structure, which is important for proper sister chromatid cohesion and release during mitosis. Cohesin, together with CTCF, is also essential to shape the architecture of the genome through its ring structure that allows for chromatin loops formation. Here, we investigated STAG2 and STAG1 functions using isogenic proficient and deficient Ewing sarcoma models. Using extensive characterization of these models by transcriptomics and epigenetics approaches, we demonstrate that STAG2 LOF has a major impact on the expression of genes associated with enhancer features, highlighting STAG2 and EWSR1-FLI1 convergent effects. Using chromatin conformation capture methods, we further show that this transcriptomic modulation results from a STAG2 LOF specific topologic alteration of the genome. This work therefore unravels a new fundamental role of STAG2 in topologic organization of the genome and in gene regulation.

This abstract is also being presented as Poster B53.

Citation Format: Didier Surdez, Sakina Zaidi, Sandrine Grossetête, Virginie Raynal, Sylvain Baulande, Véronique Hill, Olivier Delattre. STAG2 mutations alter topologic organization of the genome and cis-mediated interactions [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 PR09.
Abstract PR14: Ewing sarcoma cells exploit the IL1RAP-CTH axis to drive oxidative stress adaptation and lung metastasis
Authors: Zhang Haifeng , El-Naggar Amal M. , Cheng Hongwei , Delaidelli Alberto , Negri Gian Luca , Li Wei , Dimitrov Dimiter S. , Sorensen Poul H.B.
Published: August 2020

Metastasis is a highly inefficient process due to the high oxidative stress that cancer cells experience, such as in the circulation and at distant organs prior to colonization. How cancer cells adapt to those stressful conditions and develop anti-anoikis properties remains elusive. Here our global translatome and proteome analyses have uncovered novel signatures exploited by oncogene-transformed cells to adapt and survive anoikis-inducing stress. We reveal that EWS-ETS chimeric oncoproteins trigger the expression of the IL1RAP surface protein to alleviate oxidative stress and facilitate stress adaptation, thereby promoting lung metastasis in Ewing sarcoma. Proteomic profiling identifies CTH, a key enzyme for de novo cysteine synthesis and redox regulation, as an important functional downstream mediator of IL1RAP. Blockade of either IL1RAP or CTH, using genetic or pharmacologic approaches, renders EWS cells susceptible to oxidative stress in vitro, and dramatically mitigates primary tumor growth, local invasion and lung metastasis in mice. Importantly, high expression of this IL1RAP-CTH axis correlates with markedly decreased overall and event-free survival in EWS patients. Thus, our study has defined a novel mechanism exploited by EWS cells for metastasis, which can be a potential therapeutic target for high-risk metastatic EWS.

This abstract is also being presented as Poster B58.

Citation Format: Haifeng Zhang, Amal M. El-Naggar, Hongwei Cheng, Alberto Delaidelli, Gian Luca Negri, Wei Li, Dimiter S. Dimitrov, Poul H.B. Sorensen. Ewing sarcoma cells exploit the IL1RAP-CTH axis to drive oxidative stress adaptation and lung metastasis [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 PR14.