[Show abstract][Hide abstract] ABSTRACT: The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon-driven, functional genomic mouse model of medulloblastoma with 'humanized' in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (<5%). Whole-genome sequencing of 33 pairs of human diagnostic and post-therapy medulloblastomas demonstrated substantial genetic divergence of the dominant clone after therapy (<12% diagnostic events were retained at recurrence). In both mice and humans, the dominant clone at recurrence arose through clonal selection of a pre-existing minor clone present at diagnosis. Targeted therapy is unlikely to be effective in the absence of the target, therefore our results offer a simple, proximal, and remediable explanation for the failure of prior clinical trials of targeted therapy.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Despite significant strides in the identification and characterization of potential therapeutic targets for medulloblastoma (MB), the role of the immune system and its interplay with the tumor microenvironment within these tumors are poorly understood. To address this, we adapted two syngeneic animal models of human Sonic Hedgehog (SHH)-driven and Group 3 MB for preclinical evaluation in immunocompetent C57BL/6 mice.
Methods and results:
Multicolor flow cytometric analyses were used to phenotype and characterize immune infiltrating cells within established cerebellar tumors. We observed significantly higher percentages of dendritic cells, infiltrating lymphocytes, myeloid derived suppressor cells and tumor-associated macrophages in murine SHH model tumors compared with Group 3 tumors. However, murine Group 3 tumors had higher percentages of CD8+ PD-1+ T cells within the CD3 population. PD-1 blockade conferred superior antitumor efficacy in animals bearing intracranial Group 3 tumors compared to SHH group tumors, indicating that immunologic differences within the tumor microenvironment can be leveraged as potential targets to mediate antitumor efficacy. Further analysis of anti-PD-1 monoclonal antibody localization revealed binding to PD-1+ peripheral T cells, but not tumor infiltrating lymphocytes within the brain tumor microenvironment. Peripheral PD-1 blockade additionally resulted in a marked increase in CD3+ T cells within the tumor microenvironment.
This is the first immunologic characterization of preclinical models of molecular subtypes of MB and demonstration that response to immune checkpoint blockade differs across subtype classification. Our findings also suggest that effective anti-PD-1 blockade does not require that systemically administered antibodies penetrate the brain tumor microenvironment.
Full-text · Article · Sep 2015 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Genomic characterization of medulloblastoma has improved molecular risk classification but struggles to define functional biological processes, particularly for the most aggressive subgroups. We present here a novel proteomic approach to this problem using a reference library of stable isotope labeled medulloblastoma-specific proteins as a spike-in standard for accurate quantification of the tumor proteome. Utilizing high-resolution mass spectrometry, we quantified the tumor proteome of group 3 medulloblastoma cells and demonstrate that high-risk MYC amplified tumors can be segregated based on protein expression patterns. We cross-validated the differentially expressed protein candidates using an independent transcriptomic data set and further confirmed them in a separate cohort of medulloblastoma tissue samples to identify the most robust proteogenomic differences. Interestingly, highly expressed proteins associated with MYC-amplified tumors were significantly related to glycolytic metabolic pathways via alternative splicing of pyruvate kinase (PKM) by heterogeneous ribonucleoproteins (HNRNPs). Furthermore, when maintained under hypoxic conditions, these MYC-amplified tumors demonstrated increased viability compared to non-amplified tumors within the same subgroup. Taken together, these findings highlight the power of proteomics as an integrative platform to help prioritize genetic and molecular drivers of cancer biology and behavior.
[Show abstract][Hide abstract] ABSTRACT: Medulloblastoma (MB) is the most common malignant brain tumor in children. Genomic studies have identified four molecular
subgroups: WNT, SHH, Group 3, and Group 4. Among these, Group 3 tumors are the most aggressive and the most frequently fatal.
Aside from amplification or overexpression of the MYC oncogene (which is insufficient for tumorigenesis on its own), oncogenic
drivers for Group 3 MB remain largely unidentified. Recently, whole genome sequencing of primary MB samples identified a series
of disparate genomic structural variants restricted to Groups 3 and 4, resulting in specific and mutually exclusive activation
of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Using an orthotopic transplantation model, we show
that Gfi1 and Gfi1B can cooperate strongly with Myc to transform neural stem cells and drive MB formation in mice. The resulting
tumors are highly invasive and proliferative, and exhibit histological and molecular characteristics consistent with human
Group 3 MB. These studies suggest that Gfi1 and Gfi1B may be important drivers of tumorigenesis in Group 3 MB. Our ongoing
studies are focused on understanding the molecular mechanisms by which Gfi1 and Gfi1B contribute to tumor formation. Specifically,
we are using proteomic approaches to examine the cofactors that bind to Gfi proteins and mediate their effects, and a combination
of expression profiling and chromatin immunoprecipitation-sequencing (ChIP-Seq) to identify the molecular targets of these
transcription factors in MB. In addition, we have created conditional Gfi1-expressing mice to determine whether these proteins
are required for tumor maintenance. Finally, we are using cells from our animal models to identify therapeutic agents that
inhibit the growth of Gfi-activated tumors in vitro and in vivo. These studies will provide key insights into the biology
of Group 3 MB and help identify novel approaches to therapy.
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION: Medulloblastoma (MB) remains incurable in one third of patients despite aggressive multi-modality standard therapies.
Immunotherapy presents a promising alternative by specifically targeting cancer cells. To date, there have been no successful
immunologic applications targeting MB. Emerging evidence from integrated genomic studies has suggested MB variants arise from
deregulation of pathways affecting proliferation of progenitor cell populations within the developing cerebellum. Using total
embryonic RNA as a source of tumor rejection antigens is attractive because it can be delivered as a single vaccine, target
both known and unknown fetal proteins, and can be refined to preferentially treat distinct MB subtypes. METHODS: We have created
two transplantable, syngeneic animal MB models recapitulating human SHH and Group 3 variants to investigate the immunologic
targeting of different MB subtypes. We generated T cells specific to the developing mouse cerebellum (P5) and tested their
reactivity to target cells pulsed with total RNA from two MB subtypes and the normal brain. Immune responses were evaluated
by measuring cytokine secretion following re-stimulation of activated T cells with both normal and tumor cell targets. In
vivo antitumor efficacy was also tested in survival studies of intracranial tumor-bearing animals. RESULTS: We generated T
cells specific to the developing cerebellum in vitro, confirming the immunogenicity of developmentally regulated antigens.
Additionally, we have shown that developmental antigen-specific T cells produce high levels of Th1-type cytokines in response
to tumor cells of two immunologically distinct subtypes of MB. Interestingly, developmental antigen specific T cells do not
show cross reactivity with the normal brain or subsequent stages of the developing brain after P5. Targeting developmental
antigens also conferred a significant survival benefit in a treatment model of Group 3 tumor bearing animals. CONCLUSIONS:
Developmental antigens can safely target multiple MB subtypes with equal effectiveness compared to previously established
total tumor strategies.
[Show abstract][Hide abstract] ABSTRACT: Wnt signaling regulates self-renewal and fate commitment of stem and progenitor cells in development and homeostasis. Leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) is a co-receptor for Wnt signaling that marks highly proliferative stem and progenitor cells in many epithelial tissue types. Wnt signaling instructs neural developmental and homeostatic processes; however, Lgr5 expression in the developing and adult brain has not been characterized. Here we report that Lgr5 is expressed in the postnatal cerebellum during the maturation and synaptogenesis of cerebellar granule neurons (CGNs), processes controlled by Wnt signaling. Using a transgenic reporter mouse for in vivo Lgr5 expression analysis and lineage tracing, we reveal that Lgr5 specifically identified CGNs and was restricted temporally to the CGN maturation phase within the internal granule layer, but absent in the adult brain. Cells marked by Lgr5 were lineage restricted, post-mitotic and long-lived. The ligand for Lgr5, R-spondin, was secreted in a paracrine fashion that evolved during the maturation of CGNs, which coincided with the Lgr5 expression pattern. Our findings provide potential new insight into the critical regulation of Wnt signaling in the developing cerebellum and support a novel role for Lgr5 in the regulation of post-mitotic cells.
[Show abstract][Hide abstract] ABSTRACT: Choroid plexus tumors are the most common brain tumors in children under one year of age, accounting for up to 20% of brain
tumors in that age group. These tumors exhibit a range of malignancies, from relatively benign choroid plexus papillomas (CPPs)
to extremely aggressive choroid plexus carcinomas (CPCs), which despite intensive therapy, have a dismal prognosis. More effective
treatments for these tumors depend on animal models that can be used to study tumor biology. We have created animal models
of choroid plexus tumors by crossing mice carrying Cre-inducible alleles of Myc (LSL-MycT58A) and Cre-deletable alleles of
p53 (p53flox) to animals that express Cre recombinase in neural stem cells or progenitors (Atoh1-Cre or Blbp-Cre). The progeny
of these crosses develop choroid plexus tumors with high penetrance: activation of Myc alone results in CPPs, and concomitant
deletion of p53 accelerates tumor growth and malignancy, culminating in formation of CPCs. Histological analysis indicates
that mouse CPPs and CPCs resemble their human counterparts, and that CPCs (but not CPPs) exhibit reduced expression of normal
choroid plexus epithelial markers and loss of tight junction proteins. Gene expression profiling reveals that CPPs and CPCs
have altered expression of cell cycle regulators, and exhibit hallmarks of genomic instability and DNA damage responses. CPCs,
but not CPPs, also exhibit abnormal telomere function and have increased expression of stem cell markers and hypoxia-induced
genes. These studies provide important insight into the mechanisms of choroid plexus tumorigenesis, and yield valuable animal
models that can be used to identify novel therapies for patients with aggressive choroid plexus tumors.
[Show abstract][Hide abstract] ABSTRACT: Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and
chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoural
heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and Group 4 subgroup medulloblastomas
account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. In an effort to
uncover novel drivers in these poorly characterized subgroups, we analyzed whole-genome sequencing data derived from 137 primary
Group 3 and Group 4 medulloblastoma samples and performed a systematic, high-resolution screen for chromosomal breakpoints
recurrently targeting putative candidate genes. This comprehensive analysis of structural variants identified highly disparate
genomic structural variants, restricted to Groups 3 and 4, resulting in specific and mutually exclusive activation of the
growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Diverse mechanisms of structural variation, including
duplications, deletions, inversions, translocations, and other complex variants were observed in nearly all GFI1/GFI1B-activated
cases. Integration of genome sequencing data with sample-matched ChIP-seq data for enhancer histone marks established that
somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers,
instigating their oncogenic activity. Functional experiments performed in mice confirmed the oncogenicity of both GFI1 and
GFI1B in the context of medulloblastoma and demonstrated apparent functional synergy between both of these candidates and
MYC. These studies establish GFI1 and GFI1B as novel, highly prevalent medulloblastoma oncogenes specifically active in Group
3 and Group 4. Given their high frequencies of activation, GFI1 and GFI1B represent excellent candidates for prioritization
of molecularly targeted therapy aimed at treatment of a significant proportion of Group 3 and Group 4 medulloblastoma patients.
Moreover, our data implicates ‘enhancer hijacking’ as an efficient mechanism capable of driving oncogene activation in a childhood
[Show abstract][Hide abstract] ABSTRACT: Medulloblastoma (MB) is a highly malignant brain tumor that occurs primarily in children. Although surgery, radiation and high-dose chemotherapy have led to increased survival, many MB patients still die from their disease, and patients who survive suffer severe long-term side effects as a consequence of treatment. Thus, more effective and less toxic therapies for MB are critically important. Development of such therapies depends in part on identification of genes that are necessary for growth and survival of tumor cells. Survivin is an inhibitor of apoptosis protein that regulates cell cycle progression and resistance to apoptosis, is frequently expressed in human MB and when expressed at high levels predicts poor clinical outcome. Therefore, we hypothesized that Survivin may have a critical role in growth and survival of MB cells and that targeting it may enhance MB therapy. Here we show that Survivin is overexpressed in tumors from patched (Ptch) mutant mice, a model of Sonic hedgehog (SHH)-driven MB. Genetic deletion of survivin in Ptch mutant tumor cells significantly inhibits proliferation and causes cell cycle arrest. Treatment with small-molecule antagonists of Survivin impairs proliferation and survival of both murine and human MB cells. Finally, Survivin antagonists impede growth of MB cells in vivo. These studies highlight the importance of Survivin in SHH-driven MB, and suggest that it may represent a novel therapeutic target in patients with this disease.Oncogene advance online publication, 22 September 2014; doi:10.1038/onc.2014.304.
[Show abstract][Hide abstract] ABSTRACT: Medulloblastoma, the most common malignant childhood brain tumor, exhibits distinct molecular subtypes and cellular origins. Genetic alterations driving medulloblastoma initiation and progression remain poorly understood. Herein, we identify GNAS, encoding the G protein Gαs, as a potent tumor suppressor gene that, when expressed at low levels, defines a subset of aggressive Sonic hedgehog (SHH)-driven human medulloblastomas. Ablation of the single Gnas gene in anatomically distinct progenitors in mice is sufficient to induce Shh-associated medulloblastomas, which recapitulate their human counterparts. Gαs is highly enriched at the primary cilium of granule neuron precursors and suppresses Shh signaling by regulating both the cAMP-dependent pathway and ciliary trafficking of Hedgehog pathway components. Elevation in levels of a Gαs effector, cAMP, effectively inhibits tumor cell proliferation and progression in Gnas-ablated mice. Thus, our gain- and loss-of-function studies identify a previously unrecognized tumor suppressor function for Gαs that can be found consistently across Shh-group medulloblastomas of disparate cellular and anatomical origins, highlighting G protein modulation as a potential therapeutic avenue.
[Show abstract][Hide abstract] ABSTRACT: Genomics has illuminated the extensive intertumoural heterogeneity of medulloblastoma and identified at least four distinct molecular subgroups of the disease. Group 3 and Group 4 subgroup medulloblastomas account for the majority of pediatric cases, yet, oncogenic drivers for these subtypes remain poorly understood. Exome and genome sequencing studies have confirmed a paucity of recurrent gene-level mutations in Group 3 and Group 4, suggesting that alternative oncogenic mechanisms must account for the large fraction of cases that cannot currently be explained by single-nucleotide variants or insertions/deletions alone.