Immune checkpoint inhibitor (ICI) therapy continues to revolutionize melanoma treatment, but only a subset of patients respond. Major efforts are underway to develop minimally invasive predictive assays of ICI response. Using single-cell transcriptomics, we discovered a unique CD8 T cell blood/tumor-shared subpopulation in melanoma patients with high levels of oxidative phosphorylation (OXPHOS), the ectonucleotidases CD38 and CD39, and both exhaustion and cytotoxicity markers. We called this population with high levels of OXPHOS “CD8+ TOXPHOS cells.” We validated that higher levels of OXPHOS in tumor- and peripheral blood–derived CD8+ TOXPHOS cells correlated with ICI resistance in melanoma patients. We then developed an ICI therapy response predictive model using a transcriptomic profile of CD8+ TOXPHOS cells. This model is capable of discerning responders from nonresponders using either tumor or peripheral blood CD8 T cells with high accuracy in multiple validation cohorts. In sum, CD8+ TOXPHOS cells represent a critical immune population to assess ICI response with the potential to be a new target to improve outcomes in melanoma patients.

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SummaryNotch signaling is an important regulator of stem cell differentiation. All canonical Notch signaling is transmitted through the DNA-binding protein CSL, and hyperactivated Notch signaling is associated with tumor development; thus it may be anticipated that CSL deficiency should reduce tumor growth. In contrast, we report that genetic removal of CSL in breast tumor cells caused accelerated growth of xenografted tumors. Loss of CSL unleashed a hypoxic response during normoxic conditions, manifested by stabilization of the HIF1α protein and acquisition of a polyploid giant-cell, cancer stem cell-like, phenotype. At the transcriptome level, loss of CSL upregulated more than 1,750 genes and less than 3% of those genes were part of the Notch transcriptional signature. Collectively, this suggests that CSL exerts functions beyond serving as the central node in the Notch signaling cascade and reveals a role for CSL in tumorigenesis and regulation of the cellular hypoxic response.

Wnt/β-catenin signaling is responsible for the generation of cancer stem cells (CSCs) in many human tumors, including nasopharyngeal carcinoma (NPC). Recent studies demonstrate that Wnt or PORCN inhibitor, Wnt-C59, inhibits tumor growth in MMTV-WNT1 transgenic mice. The effect of Wnt-C59 in human tumors is not clear. In this study, the NPC cell lines investigated manifest heterogeneous responses to Wnt-C59 treatment. Wnt-C59 decreased tumor growth of SUNE1 cells in mice immediately following the administration of Wnt-C59. Mice injected with HNE1 cells did not develop visible tumors after the treatment of Wnt-C59, while control mice developed 100% tumors. Wnt-C59 inhibited stemness properties of NPC cells in a dosage-dependent manner by arresting sphere formation in both HNE1 and SUNE1 cells. Thus, Wnt-C59 has the potential to eradicate CSCs in human tumors. Active β-catenin and Axin2 proteins were strongly expressed in stromal cells surrounding growing tumors, confirming the importance of Wnt signaling activities in the microenvironment being driving forces for cell growth. These novel findings confirm the ability of Wnt-C59 to suppress Wnt-driven undifferentiated cell growth in NPC. Both anti-Wnt signaling and anti-CSC approaches are feasible strategies in cancer therapy.

BackgroundA few reports suggested that low levels of Wnt signaling might drive cell reprogramming, but these studies could not establish a clear relationship between Wnt signaling and self-renewal networks. There are ongoing debates as to whether and how the Wnt/β-catenin signaling is involved in the control of pluripotency gene networks. Additionally, whether physiological β-catenin signaling generates stem-like cells through interactions with other pathways is as yet unclear. The nasopharyngeal carcinoma HONE1 cells have low expression of β-catenin and wild-type expression of p53, which provided a possibility to study regulatory mechanism of stemness networks induced by physiological levels of Wnt signaling in these cells.ResultsIntroduction of increased β-catenin signaling, haploid expression of β-catenin under control by its natural regulators in transferred chromosome 3, resulted in activation of Wnt/β-catenin networks and dedifferentiation in HONE1 hybrid cell lines, but not in esophageal carcinoma SLMT1 hybrid cells that had high levels of endogenous β-catenin expression. HONE1 hybrid cells displayed stem cell-like properties, including enhancement of CD24+ and CD44+ populations and generation of spheres that were not observed in parental HONE1 cells. Signaling cascades were detected in HONE1 hybrid cells, including activation of p53- and RB1-mediated tumor suppressor pathways, up-regulation of Nanog-, Oct4-, Sox2-, and Klf4-mediated pluripotency networks, and altered E-cadherin expression in both in vitro and in vivo assays. qPCR array analyses further revealed interactions of physiological Wnt/β-catenin signaling with other pathways such as epithelial-mesenchymal transition, TGF-β, Activin, BMPR, FGFR2, and LIFR- and IL6ST-mediated cell self-renewal networks. Using β-catenin shRNA inhibitory assays, a dominant role for β-catenin in these cellular network activities was observed. The expression of cell surface markers such as CD9, CD24, CD44, CD90, and CD133 in generated spheres was progressively up-regulated compared to HONE1 hybrid cells. Thirty-four up-regulated components of the Wnt pathway were identified in these spheres.ConclusionsWnt/β-catenin signaling regulates self-renewal networks and plays a central role in the control of pluripotency genes, tumor suppressive pathways and expression of cancer stem cell markers. This current study provides a novel platform to investigate the interaction of physiological Wnt/β-catenin signaling with stemness transition networks.