Project description:Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell mRNA-sequencing (scRNA-seq) of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Targeting transcriptional states associated to cancer cell differentiation might unravel vulnerabilities in human CRC.
Project description:KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted therapies, immune checkpoint blockade and engineered T cells. Here, we performed a systematic high throughput combinatorial drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib. This interaction targets KRAS-directed oncogenic signaling in the aggressive and therapy resistant non-glandular mesenchymal subtype of PDAC, driven by an allelic imbalance, increased gene-dosage and expression of oncogenic KRAS. Mechanistically, the combinatorial treatment induces cell cycle arrest and cell death and initiates an interferon response. Using single cell RNA sequencing and immunophenotyping, we show that the combination therapy reprograms the immunosuppressive microenvironment and primes cytotoxic and memory T cells to infiltrate the tumors, thereby sensitizing mesenchymal PDAC to PD-L1 inhibition. This work opens new avenues to target the therapy refractory mesenchymal PDAC subtype.
Project description:KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) is highly immunosuppressive and resistant to targeted therapies, immune checkpoint blockade and engineered T cells. Here, we performed a systematic high throughput combinatorial drug screen and identified a synergistic interaction between the MEK inhibitor trametinib and the multi-kinase inhibitor nintedanib. This interaction targets KRAS-directed oncogenic signaling in the aggressive and therapy resistant non-glandular mesenchymal subtype of PDAC, driven by an allelic imbalance, increased gene-dosage and expression of oncogenic KRAS. Mechanistically, the combinatorial treatment induces cell cycle arrest and cell death and initiates an interferon response. Using single cell RNA sequencing and immunophenotyping, we show that the combination therapy reprograms the immunosuppressive microenvironment and primes cytotoxic and memory T cells to infiltrate the tumors, thereby sensitizing mesenchymal PDAC to PD-L1 inhibition. This work opens new avenues to target the therapy refractory mesenchymal PDAC subtype.
Project description:DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions, that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule and thus, single cell level that can be used to monitor disease progression and response to therapy. Nucleosome Occupancy and Methylome-Sequencing (NOMe-Seq) on IMR90 cell line and 2 GBM cell lines
Project description:Cancer precision medicine largely relies on knowledge about genetic aberrations in tumors and next-generation-sequencing studies have shown a high mutational complexity in many cancers. Although a large number of the observed mutations is believed to be not causally linked with cancer, the functional effects of many rare mutations but also of combinations of driver mutations are often unknown. Here, we perform a systems analysis of a model of EGFR-mutated non-small cell lung cancer resistant to targeted therapy that integrates whole exome sequencing, global time-course discovery phosphoproteomics and computational modeling to identify functionally relevant molecular alterations. Our approach allows for a complexity reduction from over 2,000 genetic events potentially involved in mediating resistance to only 44 phosphoproteins and 35 topologically close genetic alterations. We perform single- and combination-drug testing against the predicted phosphoproteins and discovered that targeting of HSPB1, DBNL and AKT1 showed potent anti-proliferative effects overcoming resistance against EGFR-inhibitory therapy. Our approach may therefore be used to complement mutational profiling to identify functionally relevant molecular aberrations and propose combination therapies across cancers.
