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Tumor initiation represents the first step in tumorigenesis during which normal progenitor cells undergo cell fate transition to cancer. Capturing this process as it occurs in vivo, however, remains elusive. Here we employ spatiotemporally controlled oncogene activation and tumor suppressor inhibition together with multiomics to unveil the processes underlying oral epithelial progenitor cell reprogramming into tumor initiating cells at single cell resolution. Tumor initiating cells displayed a distinct stem-like state, defined by aberrant proliferative, hypoxic, squamous differentiation, and partial epithelial to mesenchymal invasive gene programs. YAP-mediated tumor initiating cell programs included activation of oncogenic transcriptional networks and mTOR signaling, and recruitment of myeloid cells to the invasive front contributing to tumor infiltration. Tumor initiating cell transcriptional programs are conserved in human head and neck cancer and associated with poor patient survival. These findings illuminate processes underlying cancer initiation at single cell resolution, and identify candidate targets for early cancer detection and prevention., Competing Interests: Competing interests: J.S.G. has received other commercial research support from Kura Oncology, Mavupharma, Dracen, Verastem, and SpringWorks Therapeutics, and is a consultant/advisory board member for Domain Therapeutics, Pangea Therapeutics, and io9, and founder of Kadima Pharmaceuticals. The remaining authors declare no competing interests., (© 2025. The Author(s).)

Immune checkpoint blockade (ICB) targeting PD-1 and CTLA-4 has revolutionized cancer treatment. However, many cancers do not respond to ICB, prompting the search for additional strategies to achieve durable responses. G-protein-coupled receptors (GPCRs) are the most intensively studied drug targets but are underexplored in immuno-oncology. Here, we cross-integrated large singe-cell RNA-sequencing datasets from CD8 + T cells covering 19 distinct cancer types and identified an enrichment of Gα s -coupled GPCRs on exhausted CD8 + T cells. These include EP 2 , EP 4 , A 2A R, β 1 AR and β 2 AR, all of which promote T cell dysfunction. We also developed transgenic mice expressing a chemogenetic CD8-restricted Gα s -DREADD to activate CD8-restricted Gα s signaling and show that a Gα s -PKA signaling axis promotes CD8 + T cell dysfunction and immunotherapy failure. These data indicate that Gα s -GPCRs are druggable immune checkpoints that might be targeted to enhance the response to ICB immunotherapies., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The T-lineage restricted protein THEMIS has been shown to play a critical role in T cell development. THEMIS, via its distinctive CABIT domains, inhibits the catalytic activity of the tyrosine phosphatase SHP1 (PTPN6). SHP1 and THEMIS bind to the ubiquitous cytosolic adapter GRB2, and the purported formation of a tri-molecular THEMIS-GRB2-SHP1 complex facilitates inactivation of SHP1 by THEMIS. The importance of this function of GRB2 among its numerous documented activities is unclear as GRB2 binds to multiple proteins and participates in several signaling responses in thymocytes. Here, we show that similar to Themis-/- thymocytes, the primary molecular defect in GRB2-deficient thymocytes is increased catalytically active SHP1 and the developmental block in GRB2-deficient thymocytes is alleviated by deletion or inhibition of SHP1 and is exacerbated by SHP1 overexpression. Thus, the principal role of GRB2 during T cell development is to promote THEMIS-mediated inactivation of SHP1 thereby enhancing the sensitivity of TCR signaling in CD4+CD8+ thymocytes to low affinity positively selecting self-ligands., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

The T cell lineage-restricted protein THEMIS plays a critical role in T cell development at the positive selection stage. In the SHP1 activation model, THEMIS is proposed to enhance the activity of the tyrosine phosphatase SHP1 (encoded by Ptpn6 ), thereby dampening T cell antigen receptor (TCR) signaling and preventing the inappropriate negative selection of CD4 + CD8 + thymocytes by positively selecting ligands. In contrast, in the SHP1 inhibition model, THEMIS is proposed to suppress SHP1 activity, rendering CD4 + CD8 + thymocytes more sensitive to TCR signaling initiated by low-affinity ligands to promote positive selection. We sought to resolve the controversy regarding the molecular function of THEMIS. We found that the defect in positive selection in Themis -/- thymocytes was ameliorated by pharmacologic inhibition of SHP1 or by deletion of Ptpn6 and was exacerbated by SHP1 overexpression. Moreover, overexpression of SHP1 phenocopied the Themis -/- developmental defect, whereas deletion of Ptpn6 , Ptpn11 (encoding SHP2), or both did not result in a phenotype resembling that of Themis deficiency. Last, we found that thymocyte negative selection was not enhanced but was instead impaired in the absence of THEMIS. Together, these results provide evidence favoring the SHP1 inhibition model, supporting a mechanism whereby THEMIS functions to enhance the sensitivity of CD4 + CD8 + thymocytes to TCR signaling, enabling positive selection by low-affinity, self-ligand-TCR interactions.

Analysis of the transcriptional profiles of developing thymocytes has shown that T lineage commitment is associated with loss of stem cell and early progenitor gene signatures and the acquisition of T cell gene signatures. Less well understood are the epigenetic alterations that accompany or enable these transcriptional changes. Here, we show that the histone demethylase Lsd1 (Kdm1a) performs a key role in extinguishing stem/progenitor transcriptional programs in addition to key repressive gene programs during thymocyte maturation. Deletion of Lsd1 caused a block in late T cell development and resulted in overexpression of interferon response genes as well as genes regulated by the Gfi1, Bcl6, and, most prominently, Bcl11b transcriptional repressors in CD4+CD8+ thymocytes. Transcriptional overexpression in Lsd1-deficient thymocytes was not always associated with increased H3K4 trimethylation at gene promoters, indicating that Lsd1 indirectly affects the expression of many genes. Together, these results identify a critical function for Lsd1 in the epigenetic regulation of multiple repressive gene signatures during T cell development., Competing Interests: Disclosures: The authors declare no competing interests exist., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)