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PURPOSE SJMB03 (ClinicalTrials.gov identifier: NCT00085202 ) was a phase III risk-adapted trial that aimed to determine the frequency and clinical significance of biological variants and genetic alterations in medulloblastoma. PATIENTS AND METHODS Patients 3-21 years old were stratified into average-risk and high-risk treatment groups based on metastatic status and extent of resection. Medulloblastomas were molecularly classified into subgroups (Wingless [WNT], Sonic Hedgehog [SHH], group 3, and group 4) and subtypes based on DNA methylation profiles and overlaid with gene mutations from next-generation sequencing. Coprimary study end points were (1) to assess the relationship between ERBB2 protein expression in tumors and progression-free survival (PFS), and (2) to estimate the frequency of mutations associated with WNT and SHH tumors. Clinical and molecular risk factors were evaluated, and the most robust were used to model new risk-classification categories. RESULTS Three hundred thirty eligible patients with medulloblastoma were enrolled. Five-year PFS was 83.2% (95% CI, 78.4 to 88.2) for average-risk patients (n = 227) and 58.7% (95% CI, 49.8 to 69.1) for high-risk patients (n = 103). No association was found between ERBB2 status and PFS in the overall cohort ( P = .74) or when patients were stratified by clinical risk ( P = .71). Mutations in CTNNB1 (96%), DDX3X (37%), and SMARCA4 (24%) were most common in WNT tumors and PTCH1 (38%), TP53 (21%), and DDX3X (19%) in SHH tumors. Methylome profiling classified 53 WNT (17.4%), 48 SHH (15.7%), 65 group 3 (21.3%), and 139 group 4 (45.6%) tumors. A comprehensive clinicomolecular risk factor analysis identified three low-risk groups (WNT, low-risk SHH, and low-risk combined groups 3 and 4) with excellent (5-year PFS > 90%) and two very high-risk groups (high-risk SHH and high-risk combined groups 3 and 4) with poor survival (5-year PFS < 60%). CONCLUSION These results establish a new risk stratification for future medulloblastoma trials.

Nuclear hormone receptors including the estrogen receptor (ERα) and the retinoic acid receptor regulate a plethora of biological functions including reproduction, circulation and immunity. To understand how estrogen and other nuclear hormones influence antibody production, we characterized total serum antibody isotypes in female and male mice of C57BL/6J, BALB/cJ and C3H/HeJ mouse strains. Antibody levels were higher in females compared to males in all strains and there was a female preference for IgG2b production. Sex-biased patterns were influenced by vitamin levels, and by antigen specificity toward influenza virus or pneumococcus antigens. To help explain sex biases, we examined the direct effects of estrogen on immunoglobulin heavy chain sterile transcript production among purified, lipopolysaccharide-stimulated B cells. Supplemental estrogen in B-cell cultures significantly increased immunoglobulin heavy chain sterile transcripts. Chromatin immunoprecipitation analyses of activated B cells identified significant ERα binding to estrogen response elements (EREs) centered within enhancer elements of the immunoglobulin heavy chain locus, including the Eµ enhancer and hypersensitive site 1,2 (HS1,2) in the 3′ regulatory region. The ERE in HS1,2 was conserved across animal species, and in humans marked a site of polymorphism associated with the estrogen-augmented autoimmune disease, lupus. Taken together, the results highlight: (i) the important targets of ERα in regulatory regions of the immunoglobulin heavy chain locus that influence antibody production, and (ii) the complexity of mechanisms by which estrogen instructs sex-biased antibody production profiles.

Females and males differ in antibody isotype expression patterns and in immune responses to foreign- and self-antigens. For example, systemic lupus erythematosus is a condition that associates with the production of isotype-skewed anti-self antibodies, and exhibits a 9:1 female:male disease ratio. To explain differences between B cell responses in males and females, we sought to identify direct interactions of the estrogen receptor (ER) with the immunoglobulin heavy chain locus. This effort was encouraged by our previous identification of estrogen response elements (ERE) in heavy chain switch (S) regions. We conducted a full-genome chromatin immunoprecipitation analysis (ChIP-seq) using DNA from LPS-activated B cells and an ERα-specific antibody. Results revealed ER binding to a wide region of DNA, spanning sequences from the JH cluster to Cδ, with peaks in Eμ and Sμ sites. Additional peaks of ERα binding were coincident with hs1,2 and hs4 sites in the 3' regulatory region (3'RR) of the heavy chain locus. This first demonstration of direct binding of ER to key regulatory elements in the immunoglobulin locus supports our hypothesis that estrogen and other nuclear hormone receptors and ligands may directly influence antibody expression and class switch recombination (CSR). Our hypothesis encourages the conduct of new experiments to evaluate the consequences of ER binding. A better understanding of ER:DNA interactions in the immunoglobulin heavy chain locus, and respective mechanisms, may ultimately translate to better control of antibody expression, better protection against pathogens, and prevention of pathologies caused by auto-immune disease.

Sex hormones are best known for their influences on reproduction, but they also have profound influences on the immune response. Examples of sex-specific differences include: (i) the relatively poor control of influenza virus infections in males compared to females, (ii) allergic asthma, an IgE-associated hypersensitivity reaction that is exacerbated in adolescent females compared to males, and (iii) systemic lupus erythematosus, a life-threatening autoimmune disease with a 9:1 female:male bias. Here we consider how estrogen and estrogen receptor α (ERα) may influence the immune response by modifying class switch recombination (CSR) and immunoglobulin expression patterns. We focus on ERα binding to enhancers (Eμ and the 3’ regulatory region) and switch sites (Sµ and Sε) in the immunoglobulin heavy chain locus. Our preliminary data from ChIP-seq analyses of purified, activated B cells show estrogen-mediated changes in the positioning of ERα binding within and near Sμ and Sε. In the presence of estrogen, ERα is bound not only to estrogen response elements (ERE), but also to adenosine-cytidine (AC)-repeats and poly adenosine (poly A) sequences, in some cases within constant region gene introns. We propose that by binding these sites, estrogen and ERα directly participate in the DNA loop formation required for CSR. We further suggest that estrogen regulates immunoglobulin expression patterns and can thereby influence life-and-death outcomes of infection, hypersensitivity, and autoimmune disease.

Immune checkpoint blockade (ICB)-mediated rejuvenation of exhausted T-cells has emerged as a promising approach for treating various cancers and chronic infections. However, T-cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. Given that many of the impaired effector properties of terminally exhausted CD8 T-cells appear to be heritably maintained even in the absence of antigen, we investigated the role of de novo DNA methylation programming as a T-cell-intrinsic mechanism for establishing the ICB-nonresponsive state of T-cell exhaustion. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T-cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo DNA methylation programs. Blocking de novo DNA methylation in activated CD8 T-cells allowed them to retain their effector functions despite persistent stimulation during chronic viral infection. We found that these de novo epigenetic programs are not only critical for establishing T-cell exhaustion, but also restrict T-cell expansion and clonal diversity during PD-L1 blockade treatment. Moreover, these exhaustion-associated DNA methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T-cell. Therapeutic approaches to reverse these programs enhanced antitumor CD8 T-cell responses and tumor control during PD-L1 blockade therapy. These data establish de novo DNA methylation programming as a major T-cell-intrinsic barrier of ICB therapy. Targeting T-cell epigenetic programs provides great potential for fostering more powerful T-cell immunotherapies. Reference: Ghoneim, et al. Cell 2017. Citation Format: Hazem E. Ghoneim, Yiping Fan, Ardiana Moustaki, Hossam Abdelsamed, Pradyot Dash, Pranay Dogra, Robert Carter, Walid Awad, Geoff Neale, Paul Thomas, Ben Youngblood. Targeting T-cell epigenetic programs to enhance the efficacy of immune checkpoint blockade [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A189.

The physiological role of multidrug resistance protein 4 (Mrp4, Abcc4) in the testes is unknown. We found that Mrp4 is expressed primarily in mouse and human Leydig cells; however, there is no current evidence that Mrp4 regulates testosterone production. We investigated its role in Leydig cells, where testosterone production is regulated by cAMP, an intracellular messenger formed when the luteinizing hormone (LH) receptor is activated. Because Mrp4 regulates cAMP, we compared testosterone levels in Mrp4(-/-) and Mrp4(+/+) mice. Young Mrp4(-/-) mice had significantly impaired gametogenesis, reduced testicular testosterone, and disruption of Leydig cell cAMP homeostasis. Both young and adult mice had impaired testosterone production. In Mrp4(-/-) primary Leydig cells treated with LH, intracellular cAMP production was impaired and cAMP-response element-binding protein (CREB) phosphorylation was strongly attenuated. Notably, expression of CREB target genes that regulate testosterone biosynthesis was reduced in Mrp4(-/-) Leydig cells in vivo. Therefore, Mrp4 is required for normal Leydig cell testosterone production. However, adult Mrp4(-/-) mice are fertile, with a normal circulating testosterone concentration. The difference is that in 3-week-old Mrp4(-/-) mice, disruption of gonadal testosterone production up-regulates hepatic Cyp2b10, a known testosterone-metabolizing enzyme. Therefore, defective testicular testosterone production de-regulates hepatic Cyp-mediated testosterone metabolism to disrupt gametogenesis. These findings have important implications for understanding the side effects of therapeutics that disrupt Mrp4 function and are reported to alter androgen production.

Purpose Recent genome-wide screens have identified genetic variations in ARID5B associated with susceptibility to childhood acute lymphoblastic leukemia (ALL). We sought to determine the contribution of ARID5B single nucleotide polymorphisms (SNPs) to racial disparities in ALL susceptibility and treatment outcome. Patients and Methods We compared the association between ARID5B SNP genotype and ALL susceptibility in whites (> 95% European genetic ancestry; 978 cases and 1,046 controls) versus in Hispanics (> 10% Native American ancestry; 330 cases and 541 controls). We determined the relationships between ARID5B SNP genotype and ALL relapse risk in 1,605 children treated on the Children's Oncology Group (COG) P9904/9905 clinical trials. Results Among 49 ARID5B SNPs interrogated, 10 were significantly associated with ALL susceptibility in both whites and Hispanics (P < .05), with risk alleles consistently more frequent in Hispanics than in whites. rs10821936 exhibited the most significant association in both races (P = 8.4 × 10−20 in whites; P = 1 × 10−6 in Hispanics), and genotype at this SNP was highly correlated with local Native American genetic ancestry (P = 1.8 × 10−8). Multivariate analyses in Hispanics identified an additional SNP associated with ALL susceptibility independent of rs10821936. Eight ARID5B SNPs were associated with both ALL susceptibility and relapse hazard; the alleles related to higher ALL incidence were always linked to poorer treatment outcome and were more frequent in Hispanics. Conclusion ARID5B polymorphisms are important determinants of childhood ALL susceptibility and treatment outcome, and they contribute to racial disparities in this disease.

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss.

Although five-year survival rates for childhood acute lymphoblastic leukemia (ALL) are now over 80% in most industrialized countries, not all children have benefited equally from this progress. Ethnic differences in survival after childhood ALL have been reported in many clinical studies, with poorer survival observed among African Americans or those with Hispanic ethnicity when compared with European Americans or Asians. The causes of ethnic differences remain uncertain, although both genetic and non-genetic factors are likely important. Interrogating genome-wide germline SNP genotypes in an unselected large cohort of children with ALL, we observed that the component of genomic variation that co-segregated with Native American ancestry was associated with risk of relapse (P = 0.0029) even after adjusting for known prognostic factors (P = 0.017). Ancestry-related differences in relapse risk were abrogated by the addition of a single extra phase of chemotherapy, indicating that modifications to therapy can mitigate the ancestry-related risk of relapse.

Immune checkpoint blockade (ICB)-mediated rejuvenation of exhausted T cells has emerged as a promising approach for treating various cancers and chronic infections. However, T cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. Given that many of the impaired effector properties of terminally exhausted CD8 T cells appear to be heritably maintained even in the absence of antigen, we investigated the role of de novo DNA methylation programming as a cell-intrinsic mechanism for establishing the ICB-nonresponsive state of T-cell exhaustion. We report that blocking de novo DNA methylation in activated CD8 T cells allows them to retain their effector functions despite chronic stimulation during a persistent viral infection. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo methylation programs that restrict T cell expansion and clonal diversity during PD-1 blockade treatment. Moreover, these exhaustion-associated DNA methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T cells. Therapeutic approaches to reverse these programs can enhance ICB-mediated T cell rejuvenation and ultimately facilitate the control of chronic viral infections and tumor growth. These data establish de novo DNA methylation programming as a regulator of T cell exhaustion and barrier of ICB therapy. Citation Format: Hazem E. Ghoneim, Yiping Fan, Ardiana Moustaki, Hossam Abdelsamed, Pradyot Dash, Pranay Dogra, Robert Carter, Walid Awad, Geoff Neale, Paul G. Thomas, Ben Youngblood. De novo epigenetic programming restrains PD-1 blockade-mediated T cell rejuvenation [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A26.

BACKGROUND. Osteosarcoma cell lines and tumors have been shown to express epidermal growth factor receptor (EGFR) and harbor amplifications at the EGFR locus. In this study, the authors further analyzed the genomic features of EGFR in osteosarcoma tumors and investigated whether they correlate with phosphatase and tensin homolog (PTEN) expression and copy number status. METHODS. EGFR and PTEN expression was assessed by immunohistochemistry (n = 28), and copy number alterations at the EGFR and PTEN loci were surveyed using Affymetrix (Santa Clara, Calif) 50K single nucleotide polymorphism (SNP) arrays (n = 31) and fluorescence in situ hybridization (FISH) (n = 27). The EGFR tyrosine kinase domain was sequenced to survey for activating mutations (n = 34). In addition, EGFRvIII expression was assessed using reverse transcriptase polymerase chain reaction (n = 24). Results were correlated with available clinical information on 59 patients, with a median age of 14.1 years (range, 5-23years) and median follow-up of 4.4 years. RESULTS. EGFR expression was detected in the majority of osteosarcoma tumors surveyed (23 of 28; 82%). SNP arrays revealed evidence of frequent copy number gains at 7p11.2 and losses at 10q23.21. A sizeable subset (16 of 27 cases; 59%) showed gains at the EGFR locus using FISH (amplification in 4 of 27 [15%] and balanced chromosome 7 polysomy in 12 of 27 [44%]), and 12 cases showed deletions at the PTEN locus (biallelic deletions in 4 of 27 [15%] and monoallelic deletion in 9 of 27 [33%]). No activating mutations in the EGFR tyrosine kinase domain, EGFRvIII expression, or association with clinical findings were detected. CONCLUSIONS. EGFR expression and genomic gains at the EGFR locus are prevalent in osteosarcoma tumors, which also commonly harbor deletions at the PTEN locus. Cancer 2008. © 2008 American Cancer Society.

The genetic risk factors for etoposide-induced leukemia with MLL translocations remain largely unknown. To identify genetic risk factors for and novel characteristics of secondary leukemia, we profiled 116,204 single nucleotide polymorphisms (SNPs) in germline and paired leukemic cell DNA from 13 secondary leukemia/myelodysplasia cases and germline DNA from 13 matched and 156 unmatched controls, all with acute lymphoblastic leukemia treated with etoposide. We analyzed global gene expression from a partially overlapping cohort. No single locus was altered in most cases. We discovered 81 regions of loss of heterozygosity (LOH) in leukemic blasts and 309 SNPs whose allele frequencies differed in cases vs controls. Candidate genes were prioritized on the basis of genes whose SNPs or expression differentiated cases from controls or showed LOH or copy number change in germline vs paired blast DNA from the 13 cases. Three biological pathways were altered: adhesion, Wnt signaling and regulation of actin. Validation experiments using a genome scan for etoposide-induced leukemogenic MLL chimeric fusions in 15 HapMap cell lines also implicated genes involved in adhesion, a process linked to de novo leukemogenesis. Independent clinical epidemiologic and in vitro genome-wide approaches converged to identify novel pathways that may contribute to therapy-induced leukemia.

There is increasing evidence from genome-wide association studies for a strong inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children, yet the effects of protein-coding variants on ALL risk have not been systematically evaluated. Here we show a missense variant in CDKN2A associated with the development of ALL at genome-wide significance (rs3731249, P=9.4 × 10−23, odds ratio=2.23). Functional studies indicate that this hypomorphic variant results in reduced tumour suppressor function of p16INK4A, increases the susceptibility to leukaemic transformation of haematopoietic progenitor cells, and is preferentially retained in ALL tumour cells. Resequencing the CDKN2A–CDKN2B locus in 2,407 childhood ALL cases reveals 19 additional putative functional germline variants. These results provide direct functional evidence for the influence of inherited genetic variation on ALL risk, highlighting the important and complex roles of CDKN2A–CDKN2B tumour suppressors in leukaemogenesis., Genome-wide association studies indicate a strong genetic susceptibility to acute lymphoblastic leukaemia in children, though the effect on protein-coding genes is not fully understood. Here Xu and Zhang et al. identify a missense variant in CDKN2A which reduces tumour suppression.

Immune-checkpoint-blockade (ICB)-mediated rejuvenation of exhausted T cells has emerged as a promising approach for treating various cancers and chronic infections. However, T cells that become fully exhausted during prolonged antigen exposure remain refractory to ICB-mediated rejuvenation. We report that blocking de novo DNA methylation in activated CD8 T cells allows them to retain their effector functions despite chronic stimulation during a persistent viral infection. Whole-genome bisulfite sequencing of antigen-specific murine CD8 T cells at the effector and exhaustion stages of an immune response identified progressively acquired heritable de novo methylation programs that restrict T cell expansion and clonal diversity during PD-1 blockade treatment. Moreover, these exhaustion-associated DNA-methylation programs were acquired in tumor-infiltrating PD-1hi CD8 T cells, and approaches to reverse these programs improved T cell responses and tumor control during ICB. These data establish de novo DNA-methylation programming as a regulator of T cell exhaustion and barrier of ICB-mediated T cell rejuvenation.

Rhombotin-2 (RBTN-2) is a proto-oncogene only in the context of T lymphocytes. We postulated that the oncogenic effect of RBTN-2 in T cells is likely mediated by binding protein(s) with T cell-specific expression. By screening a T cell cDNA library, we identified a novel ets transcription factor that binds RBTN-2. This protein was named elf-2 because its DNA-binding domain is virtually identical to that of ets family member elf-1. Northern analyses showed similar levels of two elf-2 transcripts (3.5 kb and 3.8 kb) in all tissues except thymus. Thymocytes expressed four- to 10-fold greater amounts of the 3.5 kb transcript than other tissues. Sequence analyses of cDNA clones indicated that these transcripts encode proteins differing only at their amino termini, and likely represent alternatively spliced isoforms. These isoforms (elf-2a and elf-2b) contain identical RBTN-2 binding regions and DNA-binding domains. Elf-2b lacks a putative transactivation domain. The expression patterns suggest that RBTN-2 normally interacts equally with elf-2a and elf-2b. In contrast, when RBTN-2 is inappropriately expressed in T cells, RBTN-2 would interact predominantly with elf-2b; this interaction may lead to T cell proliferation.

Medulloblastoma is heterogeneous, being characterized by molecular subgroups that demonstrate distinct gene expression profiles. Activation of the WNT or SHH signaling pathway characterizes two of these molecular subgroups, the former associated with low-risk disease and the latter potentially targeted by novel SHH pathway inhibitors. This manuscript reports the validation of a novel diagnostic immunohistochemical method to distinguish SHH, WNT, and non-SHH/WNT tumors and details their associations with clinical, pathological and cytogenetic variables. A cohort (n = 235) of medulloblastomas from patients aged 0.4–52 years was studied for expression of four immunohistochemical markers: GAB1, β-catenin, filamin A, and YAP1. Immunoreactivity (IR) for GAB1 characterizes only SHH tumors and nuclear IR for β-catenin only WNT tumors. IRs for filamin A and YAP1 identify SHH and WNT tumors. SHH, WNT, and non-SHH/WNT tumors contributed 31, 14, and 55% to the series. All desmoplastic/nodular (D/N) medulloblastomas were SHH tumors, while most WNT tumors (94%) had a classic phenotype. Monosomy 6 was strongly associated with WNT tumors, while PTCH1 loss occurred almost exclusively among SHH tumors. MYC or MYCN amplification and chromosome 17 imbalance occurred predominantly among non-SHH/WNT tumors. Among patients aged 3–16 years and entered onto the SIOP PNET3 trial, outcome was significantly better for children with WNT tumors, when compared to SHH or non-SHH/WNT tumors, which showed similar survival curves. However, high-risk factors (M+ disease, LC/A pathology, MYC amplification) significantly influenced survival in both SHH and non-SHH/WNT groups. We describe a robust method for detecting SHH, WNT, and non-SHH/WNT molecular subgroups in formalin-fixed medulloblastoma samples. In corroborating other studies that indicate the value of combining clinical, pathological, and molecular variables in therapeutic stratification schemes for medulloblastoma, we also provide the first outcome data based on a clinical trial cohort and novel data on how molecular subgroups are distributed across the range of disease.

Recent studies of genetic abnormalities in pediatric low-grade gliomas (LGGs) have focused on activation of the ERK/MAPK pathway by KIAA1549-BRAF gene fusions in the majority of pilocytic astrocytomas (PAs) and by rare mutations in elements of the pathway across histopathologically diverse LGGs. This study reports that MYB, an oncogene not previously implicated in gliomagenesis, is activated in a diverse subset of pediatric LGGs. The study cohort comprised 57 pediatric LGGs and a comparative cohort of 59 pediatric high-grade gliomas (HGGs). The LGG cohort included 34 PAs and 23 diffuse gliomas; fibrillary astrocytomas (n = 14), oligodendroglial tumors (n = 7), and angiocentric gliomas (n = 2). MYB copy number abnormalities were disclosed using Affymetrix 6.0 SNP arrays and confirmed using interphase fluorescence in situ hybridization. Novel MYB amplifications that upregulate MYB RNA and protein expression were demonstrated in 2/14 diffuse astrocytomas. In addition, focal deletion of the terminal region of MYB was seen in 1 of 2 angiocentric gliomas (AGs). Increased expression of MYB was demonstrated by quantitative RT-PCR and immunohistochemistry. MYB upregulation at the protein level was demonstrated in a proportion of diffuse LGGs (60%), pilocytic astrocytomas (41%), and HGGs (19%), but abnormalities at the genomic level were only a feature of diffuse gliomas. Our data suggest that MYB may have a role in a subset of pediatric gliomas, through a variety of mechanisms in addition to MYB amplification and deletion.

Background Inadequate extracellular conditions can adversely affect the environment of the ER and impinge on the maturation of nascent proteins. The resultant accumulation of unfolded proteins activates a signal transduction pathway, known as the unfolded protein response, which serves primarily to protect the cell during stress and helps restore homeostasis to the ER. Principal Findings Microarray analysis of the unfolded protein response in a human medulloblastoma cell line treated with thapsigargin revealed that, in addition to known targets, a large number of proangiogenic factors were up-regulated. Real-Time PCR analyses confirmed that four of these factors, VEGFA, FGF2, angiogenin and IL8, were transcriptionally up-regulated in multiple cell lines by various ER stress inducers. Our studies on VEGFA regulation revealed that XBP-1(S), a UPR-inducible transcription factor, bound to two regions on the VEGFA promoter, and analysis of XBP-1 null mouse embryonic fibroblasts revealed that it contributes to VEGFA expression in response to ER stress. ATF4, another UPR-inducible transcription factor, also binds to the VEGFA gene, although its contribution to VEGFA transcription appeared to be fairly modest. We also found that VEGFA mRNA stability is increased in response to UPR activation, via activation of AMP kinase, demonstrating that increased mRNA levels occur at two regulatory points. In keeping with the mRNA levels, we found that VEGFA protein is secreted at levels as high as or higher than that achieved in response to hypoxia. Conclusions and Significance Our results indicate that the UPR plays a significant role in inducing positive regulators of angiogenesis. It also regulates VEGFA expression at transcriptional, post-transcriptional and post-translational levels and is likely to have widespread implications for promoting angiogenesis in response to normal physiological cues as well as in pathological conditions like cancer.

We report genetic aberrations that activate the ERK/MAP kinase pathway in 100% of posterior fossa pilocytic astrocytomas, with a high frequency of gene fusions between KIAA1549 and BRAF among these tumours. These fusions were identified from analysis of focal copy number gains at 7q34, detected using Affymetrix 250K and 6.0 SNP arrays. PCR and sequencing confirmed the presence of five KIAA1549-BRAF fusion variants, along with a single fusion between SRGAP3 and RAF1. The resulting fusion genes lack the auto-inhibitory domains of BRAF and RAF1, which are replaced in-frame by the beginning of KIAA1549 and SRGAP3, respectively, conferring constitutive kinase activity. An activating mutation of KRAS was identified in the single pilocytic astrocytoma without a BRAF or RAF1 fusion. Further fusions and activating mutations in BRAF were identified in 28% of grade II astrocytomas, highlighting the importance of the ERK/MAP kinase pathway in the development of paediatric low-grade gliomas.

Vitamin A deficiency (VAD) is a global health problem that persists in over half the countries in the world, affecting an estimated 250 million preschool children. VAD individuals suffer increased morbidity and mortality during enteric and respiratory infection, as vitamin A is an important regulator of immune system function. Previously, our lab has demonstrated that VAD mice have an impaired response to respiratory virus infection, exhibiting decreased IgA and CD8+ T cell responses to infection. Here we show that VAD mice fail to clear viral infection from the respiratory tract with the same efficiency as control vitamin A sufficient animals. We observed that prolonged viral presence can associate with an increased potential for T cell activation. As a result VAD animals experience a cytokine storm comprised of a mixed Th1/Th2 response including IFN-gamma, IL-4, IL-6 and IL-10 cytokines. Increased cytokine expression can be detected both on the transcriptional and protein level. Despite previous reports attributing VAD-related inflammation to a reduced frequency of regulatory T cells in the intestine, we observe no reduction in the number of FoxP3+ T cells in the respiratory tract of VAD animals compared to controls. Our results suggest that the increased inflammation observed during respiratory infection of VAD animals is due, at least in part, to poor IgA and CD8+ T cell responses and delayed viral clearance, and not exclusively to an absence of regulatory T cells.

The most common translocations in childhood T cell acute lymphoblastic leukemias involve the LMO2 locus on chromosome 11p13 and cause ectopic expression of the LMO2 gene in thymocytes. Transgenic mice with enforced expression of LMO2 in their thymocytes develop T cell leukemias thus demonstrating the role of LMO2 in leukemogenesis. The physiologic and leukemogenic functions of LMO2 are mediated through its transcriptional regulatory activities, but the identity of the target genes is completely unknown. In this report, we have used cDNA representational difference analysis (cDNA-RDA) to identify genes that are over-expressed and are likely to play a role in the LMO2 induced leukemias. cDNA-RDA was performed using very small amounts of mRNA pool (from 1 microg of total RNA) to reverse transcribe the cDNAs from leukemic cells or normal thymocytes. The cDNA-RDA led to the isolation of nine distinct clones that were specifically overexpressed in the leukemic cells. Sequence analysis revealed that five of the nine clones had identity or homology to known genes that are known to play a role in the pathogenesis of leukemias or other cancers. Three clones had no significant homology to any known genes and thus represent novel candidate genes. Our study demonstrates that cDNA-RDA using very small amounts of total RNA is a highly efficient method to identify novel genes that may play a role in leukemogenesis.

Abstract 878 Introduction: Treatment outcome of childhood acute lymphoblastic leukemia (ALL) has improved dramatically in the last 40 years thanks to risk-directed therapy. However, a substantial proportion of patients still experience relapse, many of whom have no known risk factors. Prior efforts to improve risk stratification have primarily focused on genetic variations of the tumor (e.g., cytogenetic abnormalities) or on assessment of early antileukemic response (e.g., upfront prednisone response, minimal residual disease [MRD] after remission induction). The role of inherited genetic variation on treatment response in children with ALL is poorly characterized. Methods: We performed a genome-wide association study to evaluate the association of genotypes at 444,044 germline SNPs with the risk of relapse in 2,535 children with newly diagnosed ALL enrolled on 5 frontline clinical trials: St. Jude Total Therapy XIIIB, XV, the Children's Oncology Group (COG) P9904, P9905, and P9906 protocols. The associations between SNP genotypes and ALL relapse were evaluated by Gray's test and by Fine and Gray's hazard regression model, adjusting for genetic ancestry and treatment regimens. To identify SNPs reproducibly associated with relapse across treatment regimens, we performed 100 rounds of discovery and replication, each round dividing patients into 2 cohorts at a 1:1 ratio. Each time, we used the discovery cohort to perform a genome-wide screen, and then filtered SNPs based on their association in the replication cohort. Finally, SNPs were prioritized on the basis of the number of times their association with relapse was replicated. Results: A total of 134 SNPs, representing 88 genomic loci, were replicated in at least 10 rounds of discovery-replication tests. Across the genome, the strongest association with relapse risk was observed at 14q22.1 in the PYGL gene (rs7142143). Each copy of the C allele at this PYGL intronic SNP (rs7142143) conferred a 3.6-fold increase in the hazard rate of relapse (P=6.7×10−9) and the association of this SNP with relapse was replicated in 79 of 100 rounds of discovery-replication tests. Of 134 relapse SNPs, 73 were associated with one or more known prognostic clinical features in childhood ALL: 32 SNPs were related to leukocyte count ≥50,000/μl at diagnosis; 19 were enriched in children older than 10 years of age; 16 were associated with hyperdiploid ALL (DNA index ≥ 1.16); and 61 were associated with ALL molecular subtype and/or lineage (MLL rearrangements, ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, or T-cell ALL). Interestingly, 110 of the 134 relapse SNPs (82%) were prognostic even among MRD-negative patients, and 133 (99%) remained significantly associated with relapse after adjusting for all known risk factors, strongly indicating the potential value of germline genetic variations in ALL risk classification. To explore the mechanisms by which SNPs might influence treatment outcome of ALL, we examined the association of the 134 relapse SNPs with four pharmacokinetic and pharmacodynamic endophenotypes in the St. Jude Total XIIIB and XV cohorts: methotrexate plasma clearance, intracellular accumulation of polyglutamated (active) methotrexate, dexamethasone plasma clearance, and asparaginase antibody levels. Fourteen of the 134 relapse SNPs were significantly associated with at least one of the four pharmacologic phenotypes in a manner consistent with a pharmacokinetically intuitive association with relapse (i.e., lower drug exposure translated into a higher risk of relapse). Conclusion: In this genome-wide association study, we systematically identified host genetic variations related to treatment outcome of childhood ALL, most of which were prognostic independent of known risk factors for relapse, and some also influenced outcome by affecting host disposition of antileukemic drugs. Disclosures: No relevant conflicts of interest to declare.

Macrophage infiltration of solid tumors correlates with poor prognosis for cancer patients. However, the tumor associated macrophage (TAM) phenotype appears complex and does not emulate the current in vitro paradigms of macrophage activation. To determine TAM phenotypes from a variety of solid tumors from distinct organ types, we analyzed global TAM gene expression profiles. > 90% of all gene expression was similar between TAMs isolated from gliomas, thymomas and spontaneously occurring neuroblastomas in mice, suggesting TAMs have a common phenotype regardless of tumor type or location. Importantly, many of the genes with the highest observed expression are involved in macrophage-mediated regulation of the inflammatory response. We have collected gene expression data from TAMs impaired in IL-1 and TLR signaling (Myd88-/-) and observed a MyD88-depedent regulation of multiple secreted proteins involved in Stat3 signaling (IL-6, IL-10, G-CSF) and T cell functional differentiation (IL-23p19, IL-12p40, IL-12p35). Harvesting macrophages from genetic mouse models, we have developed an in vitro neuroblastoma sphere/TAM tissue culture system to define the autocrine/paracrine mechanisms involved in both MyD88- and Stat3-dependent signaling networks, ultimately leading to defining their involvement in T cell differentiation and/or function. Our data will clarify the tumor/TAM relationship and potentially reveal TAM-specific targets for future therapeutic application.

Detection of minimal residual disease in acute lymphoblastic leukemia: the St Jude experience

The development of lymphoid leukemia in two children with X-SCID who underwent gene therapy was partially due to activation of the LMO-2 proto-oncogene by the retroviral LTR of the vector which inserted nearby (Hacein-Bey-Abina et al., Science 2003), highlighting the importance of vector design on the potential to activate genes near vector integration sites. As gene therapy vectors for other blood disorders are evaluated, it seems prudent to assess the safety issues regarding insertion for each particular vector in appropriate pre-clinical models. We have focused on developing γ-globin lentiviral vectors for gene therapy of the hemoglobin disorders and have documented correction of a murine model of β-thalassemia in the absence of observable adverse events (Persons et al., Blood 2003; Hanawa et al., Blood 2004). To more thoroughly evaluate the potential for vector-induced genotoxicity, we have examined whether self-inactivating (SIN) γ-globin lentiviral vectors containing erythroid-specific, β-globin locus enhancer elements can alter the expression of genes nearby the vector insertion site, as the retroviral LTR did in the X-SCID trial. To ascertain whether an integrated globin vector could influence endogenous transcriptional activity in erythroid precursors, 15 clonal spleen colony erythroblast populations (≥ 95% erythroid) containing lentiviral globin vector insertions and 15 untransduced control clones were derived from bone marrow cells of β-thalassemic mice. The transcriptional profile of each clone was determined using the Affymetrix Mouse 430A microarray (representing ~15,000 genes). Expression of 4500–6000 genes was observed in all samples. Ligation-mediated PCR was used to obtain the vector-genomic DNA junction sequences, allowing identification of vector insertion locations in 13 of the clones using the NCBI database. Of these, 6 globin vector clones had 16 genes, including N-ras, which were located within 100kb of the vector insertion site and were represented on the array. Only one gene, D3Jfr1, encoding a “cold shock” DNA binding protein and which was disrupted by an intronic vector insertion, had a change in signal value relative to the mean signal value of the controls. Real time RT-PCR confirmed a 4-fold reduction in expression of this gene. Both microarray and real time RT-PCR demonstrated that expression of N-ras was unchanged. For comparison, 15 clones with insertions of a lentiviral vector containing the MSCV retroviral LTR, were also derived, along with 10 additional mock control clones. We are currently analyzing the expression of some 116 genes that lie within 300kb of the vector insertions, relative to the mean expression level in the 25 mock transduced clones. Additionally, we have expanded analysis of the globin vector clones to evaluate changes in expression of 107 genes located within 300kb of the vector insertions. These data should prove useful to assess whether integrated SIN globin lentiviral vectors containing erythroid-specific regulatory elements have a propensity to alter transcriptional activity in the progeny of genetically modified hematopoietic stem cells, relative to vectors containing viral LTR elements.

We previously reported the isolation and functional characterization of seven adenovirus type 5 (Ad5) DNA-binding protein (DBP) point mutants (Quinn, C. O., and Kitchingman, G. R. (1986) J. Virol. 60, 653-661). Six of the seven mutants were defective in their ability to help adeno-associated virus replicate its DNA. To determine the level at which the mutations affect this function of the DBP, we analyzed several properties of the mutant proteins. All are transported to the nucleus and are post-translationally phosphorylated to the same extent. The half-lives of the proteins, measured by pulse-chase, were nearly identical to that of the wild-type DBP. The mutant DBPs were examined for their ability to bind to single-stranded DNA (ssDNA). Mutations in amino acids 322, 323, and 470 lowered the affinity of the DBP for ssDNA, while a mutation in amino acid 181 had no affect. Combinations of mutations in amino acid 470 with either 322 or 323 did not further lower the affinity of the protein for ssDNA. These data indicate that the functional defect for adeno-associated virus helper activity of the six mutants is due mainly, if not totally, to their reduced affinity for single-stranded DNA. These experiments have thus identified a functional domain of the adenovirus type 5 DBP potentially involved in DNA-protein interactions. Comparisons with temperature-sensitive DBP mutants indicate that the conserved region mutants are functionally distinct and represent a new class of DBP mutants.