Background/Objectives: The oncofetal membrane protein Claudin 6 (CLDN6) is an attractive target for T cell-based therapies. There is a lack of detailed analyses on the age-dependent expression of CLDN6 in normal tissues is lacking, which limits the expansion of CLDN6 CAR-T cell clinical trials to pediatric populations. Methods: We analyzed CLDN6 expression in extracranial solid tumors and normal tissues of children using RNA-sequencing data from over 500 pediatric solid tumor samples, qRT-PCR and immunohistochemistry (IHC) in more than 100 fresh-frozen tumor samples and, approximately, 250 formalin-fixed paraffin-embedded (FFPE) samples. We examined normal tissue expression via qRT-PCR in 32 different infant tissues and via IHC in roughly 290 tissues from donors across four age groups, as well as in fetal autopsy samples. Results: In fetal tissues, we detected CLDN6 expression primarily in the epithelial cells of several organs, including the skin, lungs, kidneys, intestinal tract, and pancreas, but not in undifferentiated blastemal cells. Postnatally, we found CLDN6-positive epithelial progenitors only during the first few weeks of life. In older-age groups, isolated clusters of CLDN6-positive progenitors were present, but in scarce quantities. In tumor tissues, we found strong and homogeneous CLDN6 expression in desmoplastic small round cell tumors and germ cell tumors. Wilms tumors demonstrated heterogeneous CLDN6 expression, notably absent in the blastemal component. Conclusions: These findings highlight an organ-specific presence of CLDN6-positive epithelial precursors that largely disappear in terminally differentiated epithelia within weeks after birth. Therefore, our data support CLDN6 as a viable therapeutic target in pediatric patients and justify their inclusion in basket studies for anti-CLDN6-based therapies.

Over several decades, cell biology research has characterized distinct forms of regulated cell death, identified master regulators such as nuclear factor kappa B (NFκB), and contributed to translating these findings in order to improve anti-cancer therapies. In the era of immunotherapy, however, the field warrants a new appraisal—the targeted induction of immunogenic cell death may offer personalized strategies to optimize anti-tumor immunity. Once again, the spotlight is on NFκB, which is not only a master regulator of cancer cell death, survival, and inflammation, but also of adaptive anti-tumor immune responses that are triggered by dying tumor cells.

Resistance to cell death remains a critical challenge in the therapy of colorectal cancer (CRC). Smac mimetics (SM) are cytotoxic agents specifically designed to maximize tumor cell killing mediated via endogenous tumor necrosis factor (TNF). In CRC, however, SM lacked clinical activity for reasons that remain incompletely understood. Here, we report that the clinically approved tyrosine kinase inhibitor Cabozantinib potently sensitizes to SM by targeting a Met-RIPK1 signaling axis in CRC. Aberrant Met hampers the activation of RIPK1, which renders CRC cells resistant to TNF/SM-mediated apoptosis and necroptosis. In turn, Cabozantinib potently inhibits Met, thereby stabilizing RIPK1 expression and converting TNF into a robust pro-death signal. SM/Cabozantinib-based regimens demonstrated anti-tumor activity in vivo, and were effective in a heterogeneous panel of patient-derived CRC of diverse molecular subtypes. In addition, we show that it is feasible to modulate between apoptosis and necroptosis to overcome therapy resistance and foster anti-tumor immunity. In summary, this work provides novel biological insight into the mechanisms of SM resistance and warrants the combinatory use of SM and Cabozantinib to enhance apoptotic and necroptotic cell death in CRC.

Necroptosis is a form of regulated cell death that has been associated with degenerative disorders, autoimmune processes, inflammatory diseases, and cancer. To better understand the biochemical mechanisms of necroptosis cell death regulation, we constructed a detailed biochemical model of tumor necrosis factor (TNF)-induced necroptosis based on known molecular interactions. Intracellular protein levels, used as model inputs, were quantified using label-free mass spectrometry, and the model was calibrated using Bayesian parameter inference to experimental protein time course data from a well-established necroptosis-executing cell line. The calibrated model accurately reproduced the dynamics of phosphorylated mixed lineage kinase domain-like protein (pMLKL), an established necroptosis reporter. A dynamical systems analysis identified four distinct modes of necroptosis signal execution, which can be distinguished based on rate constant values and the roles of the deubiquitinating enzymes A20 and CYLD in the regulation of RIP1 ubiquitination. In one case, A20 and CYLD both contribute to RIP1 deubiquitination, in another RIP1 deubiquitination is driven exclusively by CYLD, and in two modes either A20 or CYLD acts as the driver with the other enzyme, counterintuitively, inhibiting necroptosis. We also performed sensitivity analyses of initial protein concentrations and rate constants and identified potential targets for modulating necroptosis sensitivity among the biochemical events involved in RIP1 ubiquitination regulation and the decision between complex II degradation and necrosome formation. We conclude by associating numerous contrasting and, in some cases, counterintuitive experimental results reported in the literature with one or more of the model-predicted modes of necroptosis execution. Overall, we demonstrate that a consensus pathway model of TNF-induced necroptosis can provide insights into unresolved controversies regarding the molecular mechanisms driving necroptosis execution for various cell types and experimental conditions.