Abstract
Pediatric brain tumor preclinical field suffered for years from the lack of in vitro and in vivo models. With the explosion of novel therapy approaches for solid and brain tumors, including ...the immunotherapies it is essential to maximize the access to preclinical models for preclinical specificity, efficacy as well and safety. One of the many ways the Children’s Brain Tumor Network (CBTN) accelerates the pediatric brain tumor research and discovery is through support of the tumor model development program. This program focuses on the generation, characterization, and distribution of diverse models to investigators worldwide provided free of charge. Here we present the resource platform with over 150 cell lines, organoids and patient derived xenografts (PDX) developed and/or propagated at D3b at CHOP on behalf of CBTN. This platform maximizes the tumor tissue use to generate a combination of cell line, organoids and/or xenograft models grown in animals. In recent years, consortium supported over 40 requests for cells lines used in basic biology and translational studies internationally. Current efforts focusing also on supporting large-scale data generation and testing through its collaborative model (Childhood Cancer Model Atlas, Procan, National Center for Advancing Translational Sciences) to maximize the molecular information available for each tumor model essential in preclinical screenings. The generated and returned to consortia data are bound with the deidentified patient clinical information and genomic data and freely available through Kid’s First Data, Cavatica and PedcBio portals. These efforts have already attracted interest from pharma stakeholders previously not observed in pediatric brain environment. This open-source repository model is an example of a unique research partnership supported by patients and their families and built with one mission to bring fast change to kids suffering from brain tumors.
BCR/ABL kinase-positive chronic myelogenous leukemia (CML) cells display genomic instability leading to point mutations in various genes including bcr/abl and p53, eventually causing resistance to ...imatinib and malignant progression of the disease. Mismatch repair (MMR) is responsible for detecting misincorporated nucleotides, resulting in excision repair before point mutations occur and/or induction of apoptosis to avoid propagation of cells carrying excessive DNA lesions. To assess MMR activity in CML, we used an in vivo assay using the plasmid substrate containing enhanced green fluorescent protein (EGFP) gene corrupted by T:G mismatch in the start codon; therefore, MMR restores EGFP expression. The efficacy of MMR was reduced approximately 2-fold in BCR/ABL-positive cell lines and CD34(+) CML cells compared with normal counterparts. MMR was also challenged by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which generates O(6)-methylguanine and O(4)-methylthymine recognized by MMR system. Impaired MMR activity in leukemia cells was associated with better survival, accumulation of p53 but not of p73, and lack of activation of caspase 3 after MNNG treatment. In contrast, parental cells displayed accumulation of p53, p73, and activation of caspase 3, resulting in cell death. Ouabain-resistance test detecting mutations in the Na(+)/K(+) ATPase was used to investigate the effect of BCR/ABL kinase-mediated inhibition of MMR on mutagenesis. BCR/ABL-positive cells surviving the treatment with MNNG displayed approximately 15-fold higher mutation frequency than parental counterparts and predominantly G:C-->A:T and A:T-->G:C mutator phenotype typical for MNNG-induced unrepaired lesions. In conclusion, these results suggest that BCR/ABL kinase abrogates MMR activity to inhibit apoptosis and induce mutator phenotype.
Abstract
The Gift from a Child (GFAC) program was inspired by the dream of one child to donate his brain for research, recognizing the need to study tumor tissue collected at diagnosis, recurrence, ...and at the time of death. Founded by the Swifty Foundation in 2016, GFAC currently is comprised of five “Centers of Excellence” at institutions with expertise in pediatric neuro-oncology. Partnering with the Children’s Brain Tumor Network, the program’s mandate is twofold: make it possible for families to donate no matter where they live in the United States and make tissue available to scientists globally to empower discovery. In order to overcome barriers that have stifled postmortem collection in the past, GFAC has invested in Tissue Navigators - individuals at each center who coordinate all aspects of donation and communicate with families, medical providers, and laboratory scientists. In 2019 alone, GFAC coordinated 55 autopsy collections from multiple diagnosis. A key metric of the program is also capturing the global sharing and usage of each tissue sample, ensuring that tissue isn’t simply “banked” but is actively being actively used to help unravel tumor biology. To date, tissue has been used for genomic and molecular data generation, preclinical model development including cell lines and PDX models, and for novel drug screening. Together with Children’s Brain Tumor Network, the Gift from a Child program is helping to ensure the most precious gift that a family can make is used to accelerate the path to cures.
Abstract
PURPOSE: Pediatric high grade glioma (pHGG) remains a fatal disease. Increasing the number of patient derived tumor models and reagents will encourage research in pHGG and support the ...translation of basic science research discoveries. This work describes a recent multi-institution initiative to provide such a resource. METHODS: pHGG tumors with associated clinical data were prospectively collected and sequenced through the Children’s Brain Tumor Tissue Consortium (CBTTC) and Pediatric Brain Tumor Atlas (PBTA) with data deposited into PedcBioPortal for easy access and visualization. Primary tumor was dissociated and cultured to create both adherent and glioma stem cell lines analyzed by targeted and WGS/RNA sequencing. A tissue microarray (TMA) of primary pHGG tumors was created and examined by immunohistochemistry. RESULTS: The pHGG set included 81 collection events (70 patients, 54% at diagnosis, median age of 11 yrs, 52% female, 43% hemispheric). Analysis of somatic mutations and copy number alterations of known glioma genes were of expected distribution (36% H3.3, 47% TP53, 24% ATRX and 7% BRAF V600E variants). There were rare germline variants in mismatch repair genes with resultant tumor hyper-mutation. At least one established adherent and/or suspension glioma stem cell line was generated from 22 (31%) patients and validated by targeted sequencing for driver pHGG mutations. A subset of lines had WGS and RNAseq profiling which computationally clustered with their parental primary tumors. The lines’ cell biology makeup and in vivo tumor formation was also evaluated. A pHGG TMA (n=77), including 37 (53%) of the sequenced pHGG tumors, showed concordance with mutation status when stained for ATRX, H3.3 K27M and TP53. CONCLUSION: The CBTTC and PBTA have created a freely available integrated resource of characterized pHGG primary tumors, corresponding cell lines and archival fixed tissue to advance translational research for pHGG.
Abstract
The Children’s Brain Tumor Network (formerly known as Children’s Brain Tumor Consortium- CBTTC) is a global organization pioneering a model of open-science medical research to improve ...treatment and discover cures. Started in 2011, our objective was to utilize a regulatory, agreement, and governance architecture to remove existing research barriers that slowed down the pace of research and collaboration. Our network now includes 17 institutions working together to empower research. As of December 2019, over 3,600 subjects have been enrolled resulting in collection of over 45,000 specimens. Clinical data collection is longitudinal and includes medical history, diagnosis, treatment, pathology slides and reports, radiology imaging and reports, and outcome data. The tissue is collected flash-frozen, in freezing media, and fresh for the generation of pre-clinical models including cell lines. Blood is collected from the subject, with blood or saliva collected from the parents for germline comparison. Additionally, the Children’s Brain Tumor Network- Pediatric Brain Tumor Atlas has generated 952 WGS and RNAseq, 221 proteomics, with annotated clinical data. All of this data, both generated raw and processed data, has been made available broadly to the scientific community via cloud-based platforms, including the Gabriella Miller Kids First Data Resource Portal, Cavatica, and PedCbioportal. As of January 2020, we have 45 approved biospecimen requests and 80 genomic/molecular data requests. In summary, the Children’s Brain Tumor Network’s goal is to accelerate the pace of discovery by providing resources and expanding the network of scientists working towards a cure.
Abstract
Pediatric brain tumors comprise a heterogeneous molecular and histological landscape that challenges most current precision-medicine approaches. While recent large-scale efforts to ...molecularly characterize distinct histological entities have dramatically advanced the field’s capacity to classify and further define molecular subtypes, developing therapeutic and less toxic molecularly-defined clinical approaches remains a challenge. To define new approaches to meet these challenges and advance scalable, shared biospecimen- and data-resources for pediatric brain tumors, the Children’s Brain Tumor Network and Pacific Pediatric Neuro-Oncology Consortium, in partnership with the Alex’s Lemonade Stand Foundation Childhood Cancer Data Lab, launched OpenPBTA, a global open science Pediatric Brain Tumor Atlas initiative to comprehensively define the molecular landscape of pediatric brain tumors. The initiative contains multi-modal analyses of research- and clinical-trial based DNA and RNA sequences from nearly 1,000 subjects (with 1,256 tumors) along with their longitudinal clinical data. The OpenPBTA’s open science framework for analysis tests the capacity of crowd-sourced collaborative architectures to advance more rapid, iterative and integrated discovery of the underlying mechanisms of disease across pediatric brain and spinal cord tumors. Since the launch of the project, OpenPBTA has collaboratively created reproducible workflows for integrated consensus SNV, CNV, and fusion calling, enabled RNA-Seq-based classification of medulloblastoma subtypes, and more than 25 additional DNA- and RNA-based analyses. The open-science platform and associated datasets and processed results provide a continuously updated, global view of the integrated cross-disease molecular landscape of pediatric brain tumors. Such biospecimen- and clinically-linked scalable data resources provide unprecedented collaborative opportunities for precision-based, personalized therapeutic discovery and drug development with the upcoming further integration of proteomic sample data (N >300) and drug response datasets, additionally diversifying the multimodal discovery potential of crowd-sourced approaches for accelerated impact for children with brain tumors.
Abstract
BACKGROUND
Pediatric high grade glioma (pHGG) remains a fatal disease. Access to richly annotated biospecimens and patient derived tumor models will accelerate pHGG research and support ...translation of research discoveries. This work describes the pediatric high grade glioma set of the Children’s Brain Tumor Tissue Consortium (CBTTC) from the first release of the Pediatric Brain Tumor Atlas (PBTA).
METHODS
pHGG tumors with associated clinical data and imaging were prospectively collected through the CBTTC and analyzed as the Pediatric Brain Tumor Atlas (PBTA) with processed genomic data deposited into PedcBioPortal for broad access and visualization. Matched tumor was cultured to create high grade glioma cell lines analyzed by targeted and WGS and RNA-seq. A tissue microarray (TMA) of primary pHGG tumors was also created.
RESULTS
The pHGG set includes 87 collection events (73 patients, 60% at diagnosis, median age of 9 yrs, 55% female, 46% hemispheric). Operative reports, pathology reports and histology images are available for nearly all cases. Pre- and post-operative MRI images and reports are also available for a subset. Tumor WGS/RNAseq is available for 70 subjects. Analysis of somatic mutations and copy number alterations of known glioma genes were of expected distribution (36% H3.3, 47% TP53, 24% ATRX and 7% BRAFV600E variants). In our panel of pHGG, six patients (8 tumors) harbored germline mismatch repair mutations with tumor hyper-mutation. A pHGG TMA (n=77), includes 36 patient tumors with matched sequencing. At least one established glioma cell line was generated from 23 patients (32%). Unique reagents include those derived from a H3.3 G34R glioma and from tumors with mismatch repair deficiency.
CONCLUSION
The CBTTC and PBTA have created an openly available integrated resource of over 2,000 tumors, including a rich set of pHGG primary tumors, corresponding cell lines and archival fixed tissue to advance translational research for pHGG.
BCR/ABL-mediated transformation is associated with elevation of ROS which, in addition to enhancing the cytoplasmic signaling pathways, may increase the number of oxidative DNA lesions including ...DSBs. Homologous recombination (HR), involving RAD51 protein, plays an important role in the response of BCR/ABL-positive leukemia cells to numerous DSBs induced by ROS. Fanconi D2 protein (FANCD2), a member of the Fanconi protein family, is monoubiquitinated on K561 and phosphorylated by ATM on S222 in response to DSBs. The K561 monoubiquitinated form of FANCD2 interacts with RAD51 during HR, and phosphorylation of FANCD2 on S222 is important for activation of S phase checkpoint in response to DNA damage. Our studies detected an enhanced interaction between RAD51 and FANCD2 in BCR/ABL-positive leukemia cells in comparison to normal counterparts implicating the role in repair of ROS-dependent DSBs. In addition, higher levels of monoubiquitinated FANCD2 protein was detectable in CML patient cells at chronic phase and in blast crisis, and in BCR/ABL-transformed cells in comparison to non-transformed cells. This effect was reversed after inhibition of BCR/ABL kinase with STI571. Therefore, FANCD2 may play a role in BCR/ABL-mediated leukemogenesis. This speculation is supported by impaired transformation potential of the BCR/ABL kinase in FANCD2−/− murine bone marrow cells in comparison to +/+ counterparts. In addition, expression of BCR/ABL kinase, but not the kinase-deficient K1172R mutant, inhibited the proliferation rate of FANCD2−/− human lymphoblast cell line. The growth defect of BCR/ABL-positive FANCD2−/− cells was accompanied with delayed leukemogenesis in SCID mice. Growth ability of BCR/ABL-positive FANCD2−/− cells could be rescued by co-expression of the wild-type and S222A mutant of FANCD2, but not the K561R mutant. This observation suggested that K561 monoubiquitination, but not S222 phosphorylation might play an important role in BCR/ABL-mediated transformation. Since BCR/ABL cells employ RAD51-dependent HR to repair numerous DSBs induced by ROS, elevated expression of monoubiquitinated FANCD2 may facilitate this process. This hypothesis is supported by the observation that BCR/ABL-positive FANCD2−/− cells and +/+ counterparts display similar levels of ROS and oxidized DNA bases, however, the former cells accumulate more DSBs evaluated by neutral comet assay and detected by γ-H2AX foci immunostaining. This effect could be reversed by the expression of FANCD2 S222A, but not K561R mutant, again implicating HR in reparation of these DSBs. Elevated levels of ROS-mediated DSBs in BCR/ABL-positive FANCD2−/− cells did not cause any significant changes in cell cycle distribution, but resulted in discrete but persistent apoptosis. Scavenging of ROS by vitamin E and N-acetylcysteine reduced the number of DSBs and eliminated the growth defect in BCR/ABL-positive FANCD2−/− cells without affecting their +/+ counterparts. In conclusion we hypothesize that monoubiquitination of FANCD2 may play a role in BCR/ABL-dependent leukemogenesis, probably due to its ability to interact with RAD51 and facilitate HR repair of an excess of spontaneous DSBs induced by ROS.
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