Pascal Hunold

Most neuroendocrine cancers lack caspase 8 protein expression. While this feature was thought to facilitate escape from extrinsic apoptosis, its cancer-regulatory function has remained unexplored. Here, we devise a mouse model of small cell lung cancer (SCLC) recapitulating the lack of expression of caspase 8 seen in humans and uncover an unexpected role for necroptosis-fueled pre-tumoral inflammation resulting in reprogramming towards a neuronal progenitor cell-like state and increased metastatic disease. Notably, transcriptional signatures of this cellular state are enriched in relapsed and metastatic human SCLC. Mechanistically, caspase 8 loss within the pre-tumoral niche promotes inflammation marked by increased recruitment of regulatory T cells (Tregs) which are responsible for the promotion of metastatic disease. Importantly, inactivation of the necroptosis executioner MLKL reverses pre-tumoral inflammation, decreases metastasis as well as neuronal-like reprogramming. Taken together, our findings suggest that pre-tumoral inflammatory cell death contributes to neuronal progenitor mimicry, immunosuppression and increased metastasis in SCLC.

Small cell lung cancer (SCLC) is a highly aggressive type of lung cancer, characterized by rapid proliferation, early metastatic spread, frequent early relapse and a high mortality rate ^1–3 . Recent evidence has suggested that innervation has an important role in the development and progression of several types of cancer ^4,5 . Cancer-to-neuron synapses have been reported in gliomas ^6,7 , but whether peripheral tumours can form such structures is unknown. Here we show that SCLC cells can form functional synapses and receive synaptic transmission. Using in vivo insertional mutagenesis screening in conjunction with cross-species genomic and transcriptomic validation, we identified neuronal, synaptic and glutamatergic signalling gene sets in mouse and human SCLC. Further experiments revealed the ability of SCLC cells to form synaptic structures with neurons in vitro and in vivo. Electrophysiology and optogenetic experiments confirmed that cancer cells can receive NMDA receptor- and GABA _A receptor-mediated synaptic inputs. Fitting with a potential oncogenic role of neuron–SCLC interactions, we showed that SCLC cells derive a proliferation advantage when co-cultured with vagal sensory or cortical neurons. Moreover, inhibition of glutamate signalling had therapeutic efficacy in an autochthonous mouse model of SCLC. Therefore, following malignant transformation, SCLC cells seem to hijack synaptic signalling to promote tumour growth, thereby exposing a new route for therapeutic intervention.

Systematic discovery of transcription factor (TF) landscapes in low-input samples and at single cell level is a major challenge in the fields of molecular biology, genetics, and epigenetics. Here, we present cleavage under Dyna mic targets and Tag mentation (DynaTag), enabling robust mapping of TF-DNA interactions using a physiological salt solution during sample preparation. DynaTag uncovers occupancy alterations for 15 TFs in stem cell and cancer tissue models. We highlight changes in TF-DNA binding for NANOG, MYC, and OCT4, during stem-cell differentiation, at both bulk and single-cell resolutions. DynaTag surpasses CUT&RUN and ChIP-seq in signal-to-background ratio and resolution. Furthermore, using tumours of a small cell lung cancer model derived from a single female donor, DynaTag reveals increased chromatin occupancy of FOXA1, MYC, and the mutant p53 R248Q at enriched gene pathways (e.g. epithelial-mesenchymal transition), following chemotherapy treatment. Collectively, we believe that DynaTag represents a significant technological advancement, facilitating precise characterization of TF landscapes across diverse biological systems and complex models.

The mutational landscape of TP53 , a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy.

Summary: Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit EZH2 stimulates its activity by an unknown mechanism. Here, we show that PRC2 forms a dimer on chromatin in which an inactive, automethylated PRC2 protomer is the allosteric activator of a second PRC2 that is poised to methylate H3 of a substrate nucleosome. Functional assays support our model of allosteric trans -autoactivation via EED, suggesting a novel mechanism mediating context- dependent activation of PRC2. Our work showcases the molecular mechanism of auto- modification coupled dimerization in the regulation of chromatin modifying complexes.