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Radiotherapy is widely regarded as the primary therapeutic modality for nasopharyngeal cancer (NPC). Studies have shown that cancer cells with high resistance to radiation, known as radioresistant cancer cells, may cause residual illness, which in turn might contribute to the occurrence of cancer recurrence and metastasis. It has been shown that cancer stem-like cells (CSCs) exhibit resistance to radiation therapy. In the present study, fractionated doses of radiation-induced epithelial-mesenchymal transition (EMT) and ALDH+ CSCs phenotype of NPC tumor spheroids. Furthermore, it has been shown that cells with elevated ALDH activity have increased resistance to the effects of fractionated irradiation. Nuclear factor erythroid-2-related factor 2 (Nrf2) plays a pivotal role in regulating cellular antioxidant systems. A large body of evidence suggests that Nrf2 plays a significant role in the development of radioresistance in cancer. The authors’ research revealed that the application of fractionated irradiation resulted in a decline in Nrf2-dependent reactive oxygen species (ROS) levels, thereby mitigating DNA damage in ALDH+ stem-like NPC cells. In addition, immunofluorescence analysis revealed that subsequent to the process of fractionated irradiation of ALDH+ cells, activated Nrf2 was predominantly localized inside the nucleus. Immunofluorescent analysis also revealed that the presence of the nuclear Nrf2+/NQO1+/ALDH1+ axis might potentially serve as an indicator of poor prognosis and resistance to radiotherapy in patients with NPC. Thus, the authors’ findings strongly suggest that the radioresistance of ALDH-positive NPC CSCs to fractionated irradiation is regulated by nuclear Nrf2 accumulation. Nrf2 exerts its effects through the downstream effector NQO1/ALDH1, which depends on ROS attenuation.

Natural killer (NK) cells are frontline defenders against cancer and are capable of recognizing and eliminating tumor cells without prior sensitization or antigen presentation. Due to their unique HLA mismatch tolerance, they are ideal for adoptive cell therapy (ACT) because of their ability to minimize graft-versus-host-disease risk. The therapeutic efficacy of NK cells is limited in part by inhibitory immune checkpoint receptors, which are upregulated upon interaction with cancer cells and the tumor microenvironment. Overexpression of inhibitory receptors reduces NK cell-mediated cytotoxicity by impairing the ability of NK cells to secrete effector cytokines and cytotoxic granules. T-cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), a well-known checkpoint receptor involved in T-cell exhaustion, has recently been implicated in the exhaustion of NK cells. Overcoming TIGIT-mediated inhibition of NK cells may allow for a more potent antitumor response following ACT. Here, we describe a novel approach to TIGIT inhibition using self-delivering RNAi compounds (INTASYL™) that incorporates the features of RNAi and antisense technology. INTASYL compounds demonstrate potent activity and stability, are rapidly and efficiently taken up by cells, and can be easily incorporated into cell product manufacturing. INTASYL PH-804, which targets TIGIT, suppresses TIGIT mRNA and protein expression in NK cells, resulting in increased cytotoxic capacity and enhanced tumor cell killing in vitro. Delivering PH-804 to NK cells before ACT has emerged as a promising strategy to counter TIGIT inhibition, thereby improving the antitumor response. This approach offers the potential for more potent off-the-shelf products for adoptive cell therapy, particularly for hematological malignancies. The online version contains supplementary material available at 10.1007/s00262-024-03835-x.

Mutation of tet methylcytosine dioxygenase 2 (encoded by TET2 ) drives myeloid malignancy initiation and progression 1 – 3 . TET2 deficiency is known to cause a globally opened chromatin state and activation of genes contributing to aberrant haematopoietic stem cell self-renewal 4 , 5 . However, the open chromatin observed in TET2-deficient mouse embryonic stem cells, leukaemic cells and haematopoietic stem and progenitor cells 5 is inconsistent with the designated role of DNA 5-methylcytosine oxidation of TET2. Here we show that chromatin-associated retrotransposon RNA 5-methylcytosine (m 5 C) can be recognized by the methyl-CpG-binding-domain protein MBD6, which guides deubiquitination of nearby monoubiquitinated Lys119 of histone H2A (H2AK119ub) to promote an open chromatin state. TET2 oxidizes m 5 C and antagonizes this MBD6-dependent H2AK119ub deubiquitination. TET2 depletion thereby leads to globally decreased H2AK119ub, more open chromatin and increased transcription in stem cells. TET2- mutant human leukaemia becomes dependent on this gene activation pathway, with MBD6 depletion selectively blocking proliferation of TET2 -mutant leukaemic cells and largely reversing the haematopoiesis defects caused by Tet2 loss in mouse models. Together, our findings reveal a chromatin regulation pathway by TET2 through retrotransposon RNA m 5 C oxidation and identify the downstream MBD6 protein as a feasible target for developing therapies specific against TET2 mutant malignancies. Subject terms: Molecular biology, Chromatin

Head and neck squamous cell carcinoma (HNSCC) is a highly malignant disease with high death rates that have remained substantially unaltered for decades. Therefore, new treatment approaches are urgently needed. Human papillomavirus-negative tumors harbor areas of terminally differentiated tissue that are characterized by cornification. Dissecting this intrinsic ability of HNSCC cells to irreversibly differentiate into non-malignant cells may have tumor-targeting potential. We modeled the cornification of HNSCC cells in a primary spheroid model and analyzed the mechanisms underlying differentiation by ATAC-seq and RNA-seq. Results were verified by immunofluorescence using human HNSCC tissue of distinct anatomical locations. HNSCC cell differentiation was accompanied by cell adhesion, proliferation stop, diminished tumor-initiating potential in immunodeficient mice, and activation of a wound-healing-associated signaling program. Small promoter accessibility increased despite overall chromatin closure. Differentiating cells upregulated KRT17 and cornification markers. Although KRT17 represents a basal stem cell marker in normal mucosa, we confirm KRT17 to represent an early differentiation marker in HNSCC tissue. Cornification was frequently found surrounding necrotic areas in human tumors, indicating an involvement of pro-inflammatory stimuli. Indeed, inflammatory mediators activated the differentiation program in primary HNSCC cells. In HNSCC tissue, distinct cell differentiation states were found to create a common tissue architecture in normal mucosa and HNSCCs. Our data demonstrate a loss of cell malignancy upon faithful HNSCC cell differentiation, indicating that targeted differentiation approaches may be therapeutically valuable. Moreover, we describe KRT17 to be a candidate biomarker for HNSCC cell differentiation and early tumor detection. Subject terms: Cancer stem cells, Oral cancer

Immune surveillance by cytotoxic T cells eliminates tumor cells and cells infected by intracellular pathogens. This process relies on the presentation of antigenic peptides by Major Histocompatibility Complex class I (MHC-I) at the cell surface. The loading of these peptides onto MHC-I depends on the peptide loading complex (PLC) at the endoplasmic reticulum (ER). Here, we uncovered that MHC-I antigen presentation is regulated by ER-associated degradation (ERAD), a protein quality control process essential to clear misfolded and unassembled proteins. An unbiased proteomics screen identified the PLC component Tapasin, essential for peptide loading onto MHC-I, as a substrate of the RNF185/Membralin ERAD complex. Loss of RNF185/Membralin resulted in elevated Tapasin steady state levels and increased MHC-I at the surface of professional antigen presenting cells. We further show that RNF185/Membralin ERAD complex recognizes unassembled Tapasin and limits its incorporation into PLC. These findings establish a novel mechanism controlling antigen presentation and suggest RNF185/Membralin as a potential therapeutic target to modulate immune surveillance. Subject terms: Endoplasmic reticulum, ER-associated degradation, MHC class I, Antigen-presenting cells

The emerging field of synthetic morphogenesis implements synthetic biology tools to investigate the minimal cellular processes sufficient for orchestrating key developmental events. As the field continues to grow, there is a need for new tools that enable scientists to uncover nuances in the molecular mechanisms driving cell fate patterning that emerge during morphogenesis. Here, we present a platform that combines cell engineering with biomaterial design to potentiate artificial signaling in pluripotent stem cells (PSCs). This platform, referred to as PSC-MATRIX, extends the use of programmable biomaterials to PSCs competent to activate morphogen production through orthogonal signaling, giving rise to the opportunity to probe developmental events by initiating morphogenetic programs in a spatially constrained manner through non-native signaling channels. We show that the PSC-MATRIX platform enables temporal and spatial control of transgene expression in response to bulk, soluble inputs in synthetic Notch (synNotch)-engineered human PSCs for an extended culture of up to 11 days. Furthermore, we used PSC-MATRIX to regulate multiple differentiation events via material-mediated artificial signaling in engineered PSCs using the orthogonal ligand green fluorescent protein, highlighting the potential of this platform for probing and guiding fate acquisition. Overall, this platform offers a synthetic approach to interrogate the molecular mechanisms driving PSC differentiation that could be applied to a variety of differentiation protocols.

Head and neck squamous cell carcinoma (HNSCC) presents a significant challenge worldwide due to its aggressiveness and high recurrence rates post-treatment, often linked to cancer stem cells (CSCs). Melatonin shows promise as a potent tumor suppressor; however, the effects of melatonin on CSCs remain unclear, and the development of models that closely resemble tumor heterogeneity could help to better understand the effects of this molecule. This study developed a tumor scaffold based on patient fibroblast-derived decellularized extracellular matrix that mimics the HNSCC microenvironment. Our study investigates the antitumoral effects of melatonin within this context. We validated its strong antiproliferative effect on HNSCC CSCs and the reduction of tumor invasion and migration markers, even in a strongly chemoprotective environment, as it is required to increase the minimum doses necessary to impact tumor viability compared to the non-scaffolded tumorspheres culture. Moreover, melatonin exhibited no cytotoxic effects on healthy cells co-cultured in the tumor hydrogel. This scaffold-based platform allows an in vitro study closer to HNSCC tumor reality, including CSCs, stromal component, and a biomimetic matrix, providing a new valuable research tool in precision oncology.

Fatty acid synthase (FASN)-catalyzed endogenous lipogenesis is a hallmark of cancer metabolism. However, whether FASN is an intrinsic mechanism of tumor cell defense against T cell immunity remains unexplored. To test this hypothesis, here we combined bioinformatic analysis of the FASN-related immune cell landscape, real-time assessment of cell-based immunotherapy efficacy in CRISPR/Cas9-based FASN gene knockout ( FASN KO ) cell models, and mathematical and mechanistic evaluation of FASN-driven immunoresistance. FASN expression negatively correlates with infiltrating immune cells associated with cancer suppression, cytolytic activity signatures, and HLA-I expression. Cancer cells engineered to carry a loss-of-function mutation in FASN exhibit an enhanced cytolytic response and an accelerated extinction kinetics upon interaction with cytokine-activated T cells. Depletion of FASN results in reduced carrying capacity, accompanied by the suppression of mitochondrial OXPHOS and strong downregulation of electron transport chain complexes. Targeted FASN depletion primes cancer cells for mitochondrial apoptosis as it synergizes with BCL-2/BCL-X L -targeting BH3 mimetics to render cancer cells more susceptible to T-cell-mediated killing. FASN depletion prevents adaptive induction of PD-L1 in response to interferon-gamma and reduces constitutive overexpression of PD-L1 by abolishing PD-L1 post-translational palmitoylation. FASN is a novel tumor cell-intrinsic metabolic checkpoint that restricts T cell immunity and may be exploited to improve the efficacy of T cell-based immunotherapy. Subject terms: Cancer metabolism, Oncogenesis