Anomalous uptake and circulatory characteristics of the plant-based small RNA MIR2911

Inconsistent detection of plant-based dietary small RNAs in circulation has thwarted the use of dietary RNA therapeutics. Here we demonstrate mice consuming diets rich in vegetables displayed enhanced serum levels of the plant specific small RNA MIR2911. Differential centrifugation, size-exclusion chromatography, and proteinase K treatment of plant extracts suggest this RNA resides within a proteinase K-sensitive complex. Plant derived MIR2911 was more bioavailable than the synthetic RNA. Furthermore, MIR2911 exhibited unusual digestive stability compared with other synthetic plant microRNAs. The characteristics of circulating MIR2911 were also unusual as it was not associated with exosomes and fractionated as a soluble complex that was insensitive to proteinase K treatment, consistent with MIR2911 being stabilized by modifications conferred by the host. These results indicate that intrinsic stability and plant-based modifications orchestrate consumer uptake of this anomalous plant based small RNA and invite revisiting plant-based microRNA therapeutic approaches

Immunotherapeutic Targeting Of Lineage Restricted Oncoproteins In Immunogenically Silent Tumors

Recent breakthroughs in biotechnology and immunology have allowed the immune system to be harnessed in the cure of some cancers. Thus far clinical successes using immunotherapy have largely been limited to liquid tumors and solid tumors bearing high mutational burden, while the majority of cancers currently remain poor candidates for these therapies. Here we demonstrate a proof-of-principle in addressing one of the main bottlenecks currently impeding widespread application of immunotherapy, the paucity of tumor specific targets in low mutational solid tumors, and the engineering of T cells to recognize tumor-specific self-antigens. Using neuroblastoma, an aggressive, low mutational, cold tumor, we developed a process combining proteomics of MHC-presented antigens with computational methods and genomic/transcriptomic analyses of normal and tumor tissues, to identify and prioritize tumor-specific antigens derived from lineage restricted oncogenes crucial to tumor biology (derived from PHOX2B, IGFBPL1, HMX1, TH, CHRNA3, and GFRA2). We then developed methodologies for engineering CAR and TCR receptors, using phage display and single-cell sequencing of functionally-expanded antigen-specific T cells, respectively. Using these methods, we demonstrate specific binding of CAR and TCR receptors to tumor antigens and potent killing of neuroblastoma cells using engineered T cells. We describe new methods of multiplexing targets for accelerated T cell engineering and propose a vision for personalized target discovery and T cell engineering. Additionally, we explore the ability of the immune system to recognize and eliminate neoantigens arising from early cancer driver genes. We developed a model of immunoediting using TCGA data to measure the underrepresentation of HLA alleles in tumors predicted to present neoantigens as a measure of immunoediting. We show that the most common tumor driver mutations such as KRAS G12D and BRAF V600E are immunogenically silent, such that peptides derived from these mutations are not presented on HLA in the vast majority of the population, thus conferring additional evolutionary advantages to these mutations in the evasion of the immune system in addition to their oncogenic properties. We show that HLA alleles offer varying degrees of immunogenic protection against cancer, and thus propose HLA as a factor contributing to cancer susceptibility

Immunotherapeutic Targeting of Lineage Restricted Oncoproteins in Immunogenically Silent Tumors

Recent breakthroughs in biotechnology and immunology have allowed the immune system to be harnessed in the cure of some cancers. Thus far clinical successes using immunotherapy have largely been limited to liquid tumors and solid tumors bearing high mutational burden, while the majority of cancers currently remain poor candidates for these therapies. Here we demonstrate a proof-of-principle in addressing one of the main bottlenecks currently impeding widespread application of immunotherapy, the paucity of tumor specific targets in low mutational solid tumors, and the engineering of T cells to recognize tumor-specific self-antigens. Using neuroblastoma, an aggressive, low mutational, cold tumor, we developed a process combining proteomics of MHC-presented antigens with computational methods and genomic/transcriptomic analyses of normal and tumor tissues, to identify and prioritize tumor-specific antigens derived from lineage restricted oncogenes crucial to tumor biology (derived from PHOX2B, IGFBPL1, HMX1, TH, CHRNA3, and GFRA2). We then developed methodologies for engineering CAR and TCR receptors, using phage display and single-cell sequencing of functionally-expanded antigen-specific T cells, respectively. Using these methods, we demonstrate specific binding of CAR and TCR receptors to tumor antigens and potent killing of neuroblastoma cells using engineered T cells. We describe new methods of multiplexing targets for accelerated T cell engineering and propose a vision for personalized target discovery and T cell engineering. Additionally, we explore the ability of the immune system to recognize and eliminate neoantigens arising from early cancer driver genes. We developed a model of immunoediting using TCGA data to measure the underrepresentation of HLA alleles in tumors predicted to present neoantigens as a measure of immunoediting. We show that the most common tumor driver mutations such as KRAS G12D and BRAF V600E are immunogenically silent, such that peptides derived from these mutations are not presented on HLA in the vast majority of the population, thus conferring additional evolutionary advantages to these mutations in the evasion of the immune system in addition to their oncogenic properties. We show that HLA alleles offer varying degrees of immunogenic protection against cancer, and thus propose HLA as a factor contributing to cancer susceptibility

Immunotherapeutic Targeting Of Lineage Restricted Oncoproteins In Immunogenically Silent Tumors

Recent breakthroughs in biotechnology and immunology have allowed the immune system to be harnessed in the cure of some cancers. Thus far clinical successes using immunotherapy have largely been limited to liquid tumors and solid tumors bearing high mutational burden, while the majority of cancers currently remain poor candidates for these therapies. Here we demonstrate a proof-of-principle in addressing one of the main bottlenecks currently impeding widespread application of immunotherapy, the paucity of tumor specific targets in low mutational solid tumors, and the engineering of T cells to recognize tumor-specific self-antigens. Using neuroblastoma, an aggressive, low mutational, cold tumor, we developed a process combining proteomics of MHC-presented antigens with computational methods and genomic/transcriptomic analyses of normal and tumor tissues, to identify and prioritize tumor-specific antigens derived from lineage restricted oncogenes crucial to tumor biology (derived from PHOX2B, IGFBPL1, HMX1, TH, CHRNA3, and GFRA2). We then developed methodologies for engineering CAR and TCR receptors, using phage display and single-cell sequencing of functionally-expanded antigen-specific T cells, respectively. Using these methods, we demonstrate specific binding of CAR and TCR receptors to tumor antigens and potent killing of neuroblastoma cells using engineered T cells. We describe new methods of multiplexing targets for accelerated T cell engineering and propose a vision for personalized target discovery and T cell engineering. Additionally, we explore the ability of the immune system to recognize and eliminate neoantigens arising from early cancer driver genes. We developed a model of immunoediting using TCGA data to measure the underrepresentation of HLA alleles in tumors predicted to present neoantigens as a measure of immunoediting. We show that the most common tumor driver mutations such as KRAS G12D and BRAF V600E are immunogenically silent, such that peptides derived from these mutations are not presented on HLA in the vast majority of the population, thus conferring additional evolutionary advantages to these mutations in the evasion of the immune system in addition to their oncogenic properties. We show that HLA alleles offer varying degrees of immunogenic protection against cancer, and thus propose HLA as a factor contributing to cancer susceptibility

A Recurrent Mutation in Anaplastic Lymphoma Kinase with Distinct Neoepitope Conformations

The identification of recurrent human leukocyte antigen (HLA) neoepitopes driving T cell responses against tumors poses a significant bottleneck in the development of approaches for precision cancer therapeutics. Here, we employ a bioinformatics method, Prediction of T Cell Epitopes for Cancer Therapy, to analyze sequencing data from neuroblastoma patients and identify a recurrent anaplastic lymphoma kinase mutation (ALK R1275Q) that leads to two high affinity neoepitopes when expressed in complex with common HLA alleles. Analysis of the X-ray structures of the two peptides bound to HLA-B*15:01 reveals drastically different conformations with measurable changes in the stability of the protein complexes, while the self-epitope is excluded from binding due to steric hindrance in the MHC groove. To evaluate the range of HLA alleles that could display the ALK neoepitopes, we used structure-based Rosetta comparative modeling calculations, which accurately predict several additional high affinity interactions and compare our results with commonly used prediction tools. Subsequent determination of the X-ray structure of an HLA-A*01:01 bound neoepitope validates atomic features seen in our Rosetta models with respect to key residues relevant for MHC stability and T cell receptor recognition. Finally, MHC tetramer staining of peripheral blood mononuclear cells from HLA-matched donors shows that the two neoepitopes are recognized by CD8+ T cells. This work provides a rational approach toward high-throughput identification and further optimization of putative neoantigen/HLA targets with desired recognition features for cancer immunotherapy

Cross-cohort analysis identifies a TEAD4-MYCN positive-feedback loop as the core regulatory element of high-risk neuroblastoma

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 MYCNAmpneuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention