Pharmacological inhibition of nonsense-mediated mRNA decay enhances anti-tumor immunity
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Lung, Skin
SUBMITTER: 
Chen Weller
LAB HEAD: Yardena Samuels
PROVIDER: PXD073256 | Pride | 2026-04-07
REPOSITORIES: Pride
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2021-12-07 | PXD019774 | Pride
Project description:Immune checkpoint inhibitor and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential for cancers with high tumor mutation burden (TMB). Here, we employed mass spectrometry (MS)-based proteogenomic large-scale profiling to identify potential immunogenic human leukocyte antigen (HLA) Class ǀ-presented peptides in both melanoma, a “hot tumor” with high TMB, and EGFR mutant lung adenocarcinoma, a “cold tumor” with low TMB. We used cell line and patient-specific databases constructed using variants identified from whole-exome sequencing, as well as a de novo search algorithm from the PEAKS search algorithm to interrogate the mass spectrometry data of the Class I immunopeptidome. We identified 12 mutant neoantigens. Several classes of tumor-associated antigen-derived peptides were also identified. We constructed a cancer germline (CG) antigen database with 285 antigens and identified 42 Class I-presented CG antigens. We identified more than 1000 post-translationally modified (PTM) peptides representing 58 different PTMs. Our results suggest that PTMs play a critical role impacting HLA-binding affinity dramatically. Finally, leveraging de novo search and an annotated lncRNA database, we developed a novel non-canonical peptide discovery pipeline to identify 44 lncRNA-derived peptides that are presented by Class I. We validated MS/MS spectra of select peptides using synthetic peptides and performed HLA Class I binding assays to demonstrate binding of select neo-peptides and lncRNA-derived peptides to Class I proteins. In summary, we provide direct evidence of HLA Class I presentation of a large number of mutant and tumor-associated peptides for potential use as vaccine or adoptive cell therapy in melanoma and EGFR mutant lung cancer.
2021-08-23 | PXD022949 | Pride
Project description:RNA stability is meticulously controlled. Here, we sought to determine if an essential post-transcriptional regulatory mechanism plays a role in pain. Nonsense-mediated decay (NMD ) safeguards against translation of mRNAs that harbor premature termination codons and controls the stability of roughly 10% of typical protein-coding mRNAs. It hinges on the activity of the conserved kinase SMG1. Both SMG1 and its target UPF1 are expressed in sensory neurons throughout the dorsal root ganglion (DRG). SMG1 protein is present in DRG neurons and their fibers in the sciatic nerve. Using high-throughput sequencing, we examined changes in mRNA abundance following inhibition of SMG1. Among the characteristics of NMD substrates, the most prominent is structure specifically in the 3′UTR. We confirmed multiple NMD stability targets in sensory neurons including ATF4. AFT4 is preferentially translated during the integrated stress response (ISR). This led us to ask if suspension of NMD induces the ISR. Indeed, blockade of NMD increases eIF2-alpha phosphorylation, a key marker of the integrated stress response. Next, we examined the effects of SMG1 inhibition on pain-associated behaviors. Peripheral inhibition of SMG1 result in mechanical hypersensitivity that persists for several days and priming to a subthreshold dose of PGE2. Priming was fully rescued by a small molecule inhibitor of the ISR. Collectively, our results indicate that suspension of NMD promotes pain through activation of the ISR.
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Project description:Runt-related transcription factor 1 (RUNX1) is a key regulator of hematopoietic differentiation. Disruption of RUNX1 in acute myeloid leukemia (AML) induces a maturation arrest and early myeloid progenitor phenotype. RUNX1 mutations occur in 10-15% of AML and are associated with poor prognosis. One-third are frameshift mutations encoding an oncogenic protein with an elongated C-terminus translated in an alternative reading frame. Here, we investigated whether the alternative reading frame of oncogenic RUNX1 can be targeted by immunotherapy. We introduced a construct with a RUNX1 frameshift mutation into EBV-B cells with common HLA class I alleles and identified 13 neopeptides by HLA-immunopeptidomics. To investigate whether these peptides are neoantigens that can be recognized by T cells, peptide-MHC tetramers were used to screen 42 healthy individuals for specific CD8 T cells. T-cell clones were isolated for 6 neopeptides in 5 HLA alleles. Three T-cell clones recognized two HLA-B*07:02 neoantigens on AML cell line SIG-M5 C9, which was edited by CRISPR/Cas9 to express a RUNX1 frameshift mutation. The T-cell receptors (TCRs) of these clones were sequenced, and analyzed after transfer to CD8 T cells. One TCR showed effective killing of SIG-M5 C9 in vitro and in immunodeficient mice. The TCR-engineered T cells also killed patient-derived AML cells with early progenitor phenotypes, including leukemic stem cells. In conclusion, we showed that the alternative reading frame created by RUNX1 frameshift mutations can be effectively targeted, demonstrating the potential relevance of TCR-based immunotherapy to treat and improve the prognosis of patients with RUNX1-mutated AML.
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Project description:Background: Patient derived organoids (PDOs) can be established from colorectal cancers as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFN) or MEK-inhibitors. Methods: Four PDOs from chemotherapy refractory and one from a treatment naïve CRC were expanded to replicates with 100 million cells each, and HLA class I and class II peptide ligands were analysed by MS. Results: We identified an average of 9,936 unique peptides per PDO which compares favourably against published immunopeptidomics studies, suggesting high sensitivity. Loss of heterozygosity of the HLA locus was associated with low peptide diversity in one PDO. Peptides from genes without detectable expression by RNA-sequencing were rarely identified by MS. Only 3 out of 612 non-silent mutations encoded for neoantigens that were detected by MS. Treatment of four PDOs with IFN upregulated HLA class I expression and qualitatively changed the immunopeptidome, with increased presentation of IFN-inducible genes. HLA class II presented peptides increased on average 16-fold with IFN treatment. MEK-inhibitor treatment showed no consistent effect on class I or II HLA expression or the peptidome. Importantly, no additional HLA class I or II presented neoantigens became detectable with any treatment. Conclusions: Only 3 out of 612 non-synonymous mutations encoded for neoantigens that were detectable by MS. Although MS has sensitivity limits and biases, and likely underestimated the true neoantigen burden, this established a lower bound of the percentage of non-silent mutations that encode for presented neoantigens, which may be as low as 0.5%. This could be a reason for the poor responses of non-hypermutated CRCs to immune checkpoint inhibitors. MEK-inhibitors recently failed to improve checkpoint-inhibitor efficacy in CRC and the observed lack of HLA upregulation or improved peptide presentation may explain this.
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Project description:Nonsense-mediated mRNA decay (NMD) is a translation-dependent mRNA turnover pathway, which degrades transcripts containing premature termination codons. The execution of NMD requires the phosphorylation of N- and C-terminal tails of the key NMD factor UPF1, which thereby serve as binding platforms for the degradation factors SMG5, SMG6 and SMG7. UPF1 phosphorylation is mediated by the kinase SMG1, whose activity is regulated by a heterodimer consisting of SMG8 and SMG9. Recent work indicated that SMG9 functions as a bridge between SMG1 and SMG8, allowing the C-terminus of SMG8 to elicits its role of stabilizing the autoinhibitory state of SMG1. Here we established SMG8 and SMG9 knockout cell lines in human colorectal adenocarcinoma cell line HCT116 via CRISPR-Cas9. In addition, we used CRISPR-Cas9 to add a FLAG-tag to the N-terminus of endogenous UPF1. With these cell lines we wanted to explore the role of SMG8 and SMG9 for SMG1 regulation and their influence on the UPF1 interactome. Furthermore, we investigated changes in the UPF1 interactome upon treatment with the SMG1 inhibitor SMG1i, which functions as an ATP-competitive inhibitor and binds to the active site of SMG1. Cells were treated with 0, 0.1 or 1 μM SMG1i for 24 h and FLAG co-IP was performed. As controls, the HCT116 wildtype cells were treated with DMSO.
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Project description:Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leukocyte antigens (HLA) on the surface of native tumor tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumor tissue samples harboring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumor-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient`s tumor and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumor material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.
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