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Differential expression and biological pathway enrichment analysis of neuroblastoma molecular subtypes

Supplementary Table 1. DNA fragments synthesized and inserted into MSCV-CD19-IRES-DsRedFP via EcoRI/BglII or BglII/XhoI digestion, and later moved into pMX-IRES-Blast via EcoRI/XhoI cloning. Supplementary Table 2. Sets of primers used for PCR amplification and Sanger sequencing of CD19 gene and the upstream -1.2Kb enhancer and promoter regions. Supplementary Table 3. Sets of primers for the analysis of CpG-Methylation status after bisulfite modification of genomic DNA. Supplementary Table 4. Sets of primers used for radioactive semiquantitative PCR analysis of CD19 mRNA isoforms. Supplementary Table 5. Sequence of the mini-gene expanding exon2 and 220nt of its flanking introns that was synthesized (Genewiz) and inserted into pBSKii+ via XhoI/NotI cloning. Supplementary Table 6. Antibodies used for pull down assays for the identification of splicing factors that bind to the CD19-int1-exon2-int2 mini-gene. Supplementary Table 7. Primary and secondary antibodies used for immunoblotting and immunoprecipitations after protein separation by SDS-PAGE and transference to PVDF membrane (N/A, not applicable). Supplementary Table 8. Pairs of oligos for semiquantitative PCR amplification of CD19 cDNA followed by visualization in agarose gel. Same primers were used for sequencing after gel purification of specific bands. Supplementary Table 9. Pairs of oligos used for Real Time-qPCR amplification of cDNA.

Supplementary Figure 1. Enhanced skipping of exons 2 and 5-6 in post-CART-19 leukemias. Supplementary Figure 2. Analysis of splicing factors involved in alternative splicing of CD19-exon2.

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module—centered around a TEAD4–MYCN positive feedback loop—emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN-amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo. Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas.Significance: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4–MYCN positive feedback loop in MYCNAmp neuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention. Cancer Discov; 8(5); 582–99. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517

Supplementary Experimental Procedures; Supplementary Figures S1-S10; Supplementary Table Legends S1-S9; Supplementary Tables S8-S9; Additional References

Purpose: Dihydrotestosterone (DHT) is an important factor in prostate cancer (PCA) genesis and disease progression. Given PCA's strong genetic component, we evaluated the possibility that variation in genes involved in DHT metabolism influence PCA risk.Experimental Design: We investigated copy number variants (CNV) and single nucleotide polymorphisms (SNP). We explored associations between CNV of uridine diphospho-glucuronosyltransferase (UGT) genes from the 2B subclass, given their prostate specificity and/or involvement in steroid metabolism and PCA risk. We also investigated associations between SNPs in genes (HSD3B1, SRD5A1/2, and AKR1C2) involved in the conversion of testosterone to DHT, and in DHT metabolism and PCA risk. The population consisted of 426 men (205 controls and 221 cases) who underwent prostate-specific antigen screening as part of a PCA early detection program in Tyrol, Austria.Results: No association between CNV in UGT2B17 and UGT2B28 and PCA risk was identified. Men carrying the AA genotype at SNP rs6428830 (HSD3B1) had an odds ratio (OR) of 2.0 [95% confidence intervals (95% CI), 1.1-4.1] compared with men with GG, and men with AG or GG versus AA in rs1691053 (SRD5A1) had an OR of 1.8 (95% CI, 1.04-3.13). Individuals carrying both risk alleles had an OR of 3.1 (95% CI, 1.4-6.7) when compared with men carrying neither (P = 0.005). Controls with the AA genotype on rs7594951 (SRD5A2) tended toward higher serum DHT levels (P = 0.03).Conclusions: This is the first study to implicate the 5α-reductase isoform 1 (SRD5A1) and PCA risk, supporting the rationale of blocking enzymatic activity of both isoforms of 5α-reductase for PCA chemoprevention. Cancer Epidemiol Biomarkers Prev; 19(1); 229–39

Supplementary Tables 1-2, Supplementary Figures 1-2 from Genetic Variation of Genes Involved in Dihydrotestosterone Metabolism and the Risk of Prostate Cancer

BACKGROUND We evaluated the combination of retifanlimab, INCAGN01876, and FSRT in patients with recurrent GBM. METHODS Phase II, single-center, 2 cohort study (A: single-arm non-surgical cohort; B, two-arm neoadjuvant/surgical cohort). Cohort A patients received pre-FSRT one-time doses of retifanlimab (500mg) and INCAGN01876 (300mg), FSRT (8 Gy x 3 fractions), and post-FSRT 28-day treatment cycles (retifanlimab, day 1; INCAGN01876, days 1, 15). Cohort B patients received pre-surgery doses of retifanlimab + INCAGN01876; subsequently, patients either went directly to resection (Sub-Arm 1) or to FSRT followed by resection (Sub-Arm 2). All patients resumed immunotherapy post-operatively. Primary endpoint: ORR in Cohort A. Data cut-off for this analysis was June 30, 2022. RESULTS Thirty-two evaluable patients: Cohort A, n=16; Cohort B, n=16 (Sub-Arm 1, n=8, Sub-Arm 2, n=8). Median follow-up time: Cohort A, 13.6 months; Cohort B, 8.8 months. Forty-four percent women, median age 64 (IQR, 55–65), 56% MGMT unmethylated. Most common grade 3/4 treatment-related AEs included cerebral edema (25%), lymphopenia (16%), and cognitive disturbance (13%). Efficacy in Cohort A: no objective responses observed, best response of stable disease achieved in 9/16 patients (56%), median PFS 3.9 months (95% CI 2.1 – 6.2 months), median OS 9.8 months (95% CI 8.3 months – not reached [NR]). Efficacy in Cohort B: median PFS NR, median OS 15.1 months (95% CI 8.5 months – NR). Median PFS and OS are longer in Cohort B Sub-Arm 2 compared to Cohort B Sub-Arm 1 (PFS, NR vs. 2.2 months, p = 0.009; OS, NR vs. 8.5 months, p=0.026). Results of tissue and blood-based immune correlative analyses will be presented. CONCLUSIONS The combination of retifanlimab, INCAGN01876, and FSRT is generally well-tolerated in patients with recurrent GBM when administered with or without surgical resection. Survival outcomes in the neoadjuvant cohort are encouraging, largely driven by patients that received neoadjuvant FSRT.

Mammals produce large quantities of mucosal and systemic antibodies that maintain the intestinal barrier, shape the intestinal microbiome and promote lifelong mutualism with commensal microbes. Here, we developed an integrated host-commensal approach combining microbial flow cytometry and 16S rRNA gene sequencing to define the population of microbes that induce mucosal and systemic antibodies with CyTOF analysis in pediatric selective Immunoglobulin A (IgA) deficient and household control siblings to determine the impacts of IgA deficiency on host cellular immune phenotype. In healthy controls, mucosal secretory IgA and IgM antibodies coat an overlapping subset of microbes, predominantly Firmicutes and Proteobacteria. Serum IgG antibodies target a similar consortium of fecal microbes, revealing connections between mucosal and systemic antibody networks. Furthermore, we find broad systemic immune dysregulation in a subset of children and mice lacking IgA, including enhanced IgG targeting of fecal microbiota, elevated levels of inflammatory and allergic cytokines and alterations in T cell activation state. Thus, IgA tunes systemic interactions between the host and commensal microbiota. Understanding how IgA affects baseline immune tone has implications for predicting and preventing autoimmune, inflammatory and allergic diseases, as well as providing improved prognostic guidance to patients with IgA deficiency. Supported by CHOP Microbiome Pilot Grant K08AI135091 Burroughs Wellcome Fund, the American Academy of Allergy, Asthma, and Immunology, the Immune Deficiency Foundation, the Primary Immune Deficiency Treatment Consortium and Chan Zuckerberg Initiative

CD8 T cells are critical for clearing infections and immune surveillance of cancers. However, in the setting of chronic viral infections and tumor growth, CD8 T cells become exhausted. T cell exhaustion (TEX) is characterized by a progressive increase of inhibitory receptor expression and concomitant diminishment of effector function. This diminished function renders the host unable to clear the virus or cancer. The TEX population is heterogenous and can be divided broadly into TEX progenitor and TEX terminal populations. TEX progenitor are self-renewing, responsive to PD-1 blockade and give rise to the later TEX subsets, including short-lived TEX terminal cells. Control of DNA demethylation is required in multiple settings for maintenance or loss of stem-like characteristics. We report that TET2, a methylcytosine dioxygenase that mediates active DNA demethylation, is required for TEX progenitors to differentiate fully into TEX terminal cells. TET2-deficiency during persistent viral infection resulted in a numerical loss of TEX terminal cells, while maintaining the TEX progenitor population. The TEX terminal cells that escaped this differentiation block retained some features of TEX progenitors, including decreased expression of several inhibitory receptors. TET2-deficient TEX progenitors responded to PD-1 blockade but were unable to establish expansion of TEX cells thought to be required for viral control. These data highlight the role of DNA methylation and TET2 in establishing TEX diversity.