Project description:To investigate the gene expression patterns in Foxg1 frameshift mutation, we performed RNAseq in P1 cortices of Q84Pfs heterozygote and littermate wild type control mice.
Project description:DNA mismatch repair deficiency (MMRD) drives microsatellite instability (MSI). Cells with MSI accumulate numerous frameshift mutations. Frameshift mutations affecting cancer-related genes may promote tumorigenesis and, therefore, are shared among independently arising MSI tumors. Consequently, such recurrent frameshift mutations can give rise to shared immunogenic frameshift peptides (FSPs) that represent ideal candidates for a vaccine against MSI cancer. Pathogenic germline variants of mismatch repair genes cause Lynch syndrome (LS), a hereditary cancer syndrome affecting approximately 20-25 million individuals worldwide. LS individuals are at high risk of developing MSI cancer. Previously, we demonstrated safety and immunogenicity of an FSP-based vaccine in a Phase I/IIa clinical trial. However, the cancer-preventive effect of FSP vaccination in the scenario of LS has not been demonstrated so far.
Project description:Lynch syndrome (LS) patients develop DNA mismatch repair deficient tumors which generate high loads of neoantigens (neoAgs), thus constituting a well-defined population that can benefit from cancer immune-interception strategies, including neoantigen-based vaccines. Using paired whole-exome sequencing and mRNAseq of colorectal cancers (CRC) (n=13) and pre-cancers (n=61) from our LS patient cohort (N=46), we performed in-silico prediction and immunogenicity ranking of highly recurrent frameshift-neoags, followed by their in-vitro validation. We described the somatic mutation landscape in all cancers and pre-cancers, and showed that mutation burden is positively correlated with neoAgs load. Furthermore, our in-vitro validation showed a 65% validation rate of our top 100 predicted neoags. Consistent with neoAgs burden, our transcriptomic results revealed increased infiltration of CD8+ and CD4+ T-cells in microsatellite unstable samples. Overall, our neoAgs catalog and all other findings, improve our understanding of cancer development in LS and guide us towards the advancement of immunoprevention vaccine strategies.
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.
Project description:This study investigates the proteomic consequences of a frameshift mutation in the NOS1 (neuronal nitric oxide synthase) gene in the context of Hirschsprung's disease. Label-free quantitative (LFQ) mass spectrometry was performed on formalin-fixed paraffin-embedded (FFPE) colon tissue sections from one NOS1-mutant patient (3 technical replicates) and three control individuals. The study comprises two experimental arms: (1) total proteome profiling of all six samples, and (2) SDS-PAGE gel-band fractionation targeting proteins at ~55 kDa and ~160 kDa (migrating and narrow segments) for the three control samples. Raw data were acquired on a Thermo Fisher Orbitrap mass spectrometer and searched against the UniProt human reference proteome (UP000005640) using MaxQuant. A total of 2,193 proteins were quantified after QC filtering. Differential analysis (proDA, limma) identified 67 significantly altered proteins (adj.P<0.05, |log₂FC|>0.5; 25 upregulated, 42 downregulated) in the NOS1 mutant versus controls. NOS1 protein itself was not detected, consistent with mutation-induced protein instability or degradation. This dataset provides the first proteomic characterization of NOS1 loss-of-function in human colon tissue and complements parallel transcriptomic (RNA-seq, Iso-Seq) and immune profiling (flow cytometry) datasets from the same NOS1 mutation cohort.