H3K36 methylation maintains cell identity by regulating opposing lineage programmes.

The epigenetic mechanisms that maintain differentiated cell states remain incompletely understood. Here we employed histone mutants to uncover a crucial role for H3K36 methylation in the maintenance of cell identities across diverse developmental contexts. Focusing on the experimental induction of pluripotency, we show that H3K36M-mediated depletion of H3K36 methylation endows fibroblasts with a plastic state poised to acquire pluripotency in nearly all cells. At a cellular level, H3K36M facilitates epithelial plasticity by rendering fibroblasts insensitive to TGFß signals. At a molecular level, H3K36M enables the decommissioning of mesenchymal enhancers and the parallel activation of epithelial/stem cell enhancers. This enhancer rewiring is Tet dependent and redirects Sox2 from promiscuous somatic to pluripotency targets. Our findings reveal a previously unappreciated dual role for H3K36 methylation in the maintenance of cell identity by integrating a crucial developmental pathway into sustained expression of cell-type-specific programmes, and by opposing the expression of alternative lineage programmes through enhancer methylation.

Uncovering transcriptomic landscape alterations of CAN-2409 in in vitro and in vivo glioma models.

Rationale: CAN-2409 is a locally delivered oncolytic therapy, which results in vaccination against the injected tumor. CAN-2409 consists of a non-replicating adenovirus armed with the Herpes virus thymidine kinase, which metabolizes ganciclovir into a phosphorylated nucleotide that is incorporated into the tumor cell's genome, thereby inflicting immunogenic cancer cell death. While CAN-2409's immunological impact has been well characterized, its effects on the tumor cells transcriptome remains unknown. We compared the transcriptomic landscape after treatment of glioblastoma models with CAN-2409 in vitro and in vivo to assess how the interplay with the tumor microenvironment influences CAN-2409-mediated transcriptome alterations. Methods: We performed RNA-Seq with CAN-2409 treated patient-derived glioma stem-like cells and tumors of C57/BL6 mice and compared KEGG pathway usage and differential gene expression focusing on immune cell and cytokine profiles. T-cell -killing assays were performed to assess candidate effectors. Results: PCA analysis showed distinct clustering of control and CAN-2409 samples under both conditions. KEGG pathway analysis revealed significant enrichment for p53 signaling and cell cycle pathway, with similar dynamics for key regulators of both pathways in vitro and in vivo, including MYC, CCNB1, PLK1 and CDC20. Selected alterations (PLK1 and CCNB1) were validated at the protein level. Cytokine expression analysis revealed upregulation of pro-inflammatory IL12a under both conditions; immune cell gene profiling showed reduction of myeloid associated genes. T-cell-killing assays showed increased killing in the presence of IL-12. Conclusion: CAN-2409 significantly alters the transcriptome both in vitro and in vivo. Comparison of pathway enrichment revealed mutual and differential utilization of pathways under both conditions, suggesting a modulating influence on the cell cycle in tumor cells, and of the tumor microenvironment on the transcriptome in vivo. IL-12 synthesis likely depends on interactions with the tumor microenvironment, and it facilitates CAN-2409 cell killing. This dataset provides potential to understand resistance mechanisms and identify potential biomarkers for future studies.

Perturbing DDR signaling enhances cytotoxic effects of local oncolytic virotherapy and modulates the immune environment in glioma.

CAN-2409 is a replication-deficient adenovirus encoding herpes simplex virus (HSV) thymidine kinase (tk) currently in clinical trials for treatment of glioblastoma. The expression of tk in transduced cancer cells results in conversion of the pro-drug ganciclovir into a toxic metabolite causing DNA damage, inducing immunogenic cell death and immune activation. We hypothesize that CAN-2409 combined with DNA-damage-response inhibitors could amplify tumor cell death, resulting in an improved response. We investigated the effects of ATR inhibitor AZD6738 in combination with CAN-2409 in vitro using cytotoxicity, cytokine, and fluorescence-activated cell sorting (FACS) assays in glioma cell lines and in vivo with an orthotopic syngeneic murine glioma model. Tumor immune infiltrates were analyzed by cytometry by time of flight (CyTOF). In vitro, we observed a significant increase in the DNA-damage marker γH2AX and decreased expression of PD-L1, pro-tumorigenic cytokines (interleukin-1ß [IL-1ß], IL-4), and ligand NKG2D after combination treatment compared with monotherapy or control. In vivo, long-term survival was increased after combination treatment (66.7%) compared with CAN-2409 (50%) and control. In a tumor re-challenge, long-term immunity after combination treatment was not improved. Our results suggest that ATR inhibition could amplify CAN-2409's efficacy in glioblastoma through increased DNA damage while having complex immunological ramifications, warranting further studies to determine the ideal conditions for maximized therapeutic benefit.

Dissecting dual roles of MyoD during lineage conversion to mature myocytes and myogenic stem cells.

The generation of myotubes from fibroblasts upon forced MyoD expression is a classic example of transcription factor-induced reprogramming. We recently discovered that additional modulation of signaling pathways with small molecules facilitates reprogramming to more primitive induced myogenic progenitor cells (iMPCs). Here, we dissected the transcriptional and epigenetic dynamics of mouse fibroblasts undergoing reprogramming to either myotubes or iMPCs using a MyoD-inducible transgenic model. Induction of MyoD in fibroblasts combined with small molecules generated Pax7+ iMPCs with high similarity to primary muscle stem cells. Analysis of intermediate stages of iMPC induction revealed that extinction of the fibroblast program preceded induction of the stem cell program. Moreover, key stem cell genes gained chromatin accessibility prior to their transcriptional activation, and these regions exhibited a marked loss of DNA methylation dependent on the Tet enzymes. In contrast, myotube generation was associated with few methylation changes, incomplete and unstable reprogramming, and an insensitivity to Tet depletion. Finally, we showed that MyoD's ability to bind to unique bHLH targets was crucial for generating iMPCs but dispensable for generating myotubes. Collectively, our analyses elucidate the role of MyoD in myogenic reprogramming and derive general principles by which transcription factors and signaling pathways cooperate to rewire cell identity.

Molecular imaging in endoscopy.

Molecular imaging focuses on the molecular signature of cells rather than morphological changes in the tissue. The need for this novel type of imaging arises from the often difficult detection and characterization especially of small and/or premalignant lesions. Molecular imaging specifically visualizes biological properties of a lesion and might thereby be able to close diagnostic gaps, e.g. when differentiating hyperplastic from neoplastic polyps or detecting the margins of intraepithelial neoplastic spread. Additionally, not only the detection and discrimination of lesions could be improved: based on the molecular features identified using molecular imaging, therapy regimens could be adjusted on the day of diagnosis to allow for personalized medicine and optimized care for each individual patient.

Molecular in vivo imaging of gastric cancer in a human-murine xenograft model: targeting epidermal growth factor receptor.

BACKGROUND: The prognosis of gastric cancer depends on early diagnosis. Targeted therapies against epidermal growth factor receptors (EGFRs) are currently emerging for the treatment of gastric cancer. OBJECTIVE: To specifically visualize gastric cancer by using monoclonal antibodies targeting EGFR1 as molecular probes for in vivo molecular confocal laser endomicroscopy (mCLE) in a human-murine xenograft model. DESIGN: Prospective in vivo animal study. SETTING: Animal laboratory. INTERVENTIONS: Human gastric carcinoma xenografts were examined in 26 nude mice by using mCLE after injection of fluorescently labeled antibodies. Nine mice received low-dose anti-EGFR1 antibodies, 7 mice cetuximab, and 7 control mice isotype antibodies. Three mice were screened for autofluorescence without injection. Macroscopic fluorescence was evaluated in 2 additional mice. MAIN OUTCOME MEASUREMENTS: Molecular imaging of gastric cancer with confocal laser endomicroscopy. RESULTS: Fluorescence intensity in the anti-EGFR1 (P = .0145) and cetuximab group (P = .0047) was significantly higher than in isotype control mice. The same protocol allowed macroscopic fluorescence detection of tumor xenografts. LIMITATIONS: Animal model. CONCLUSIONS: In vivo microscopic and macroscopic molecular imaging of gastric cancer is feasible in a human-murine xenograft model with both diagnostic and therapeutic antibodies targeting EGFR1. In perspective, mCLE could help diagnose and molecularly characterize gastric cancer during ongoing gastroscopy and may even assist in the prediction of response to therapy.