Abstract In humans, tissue homeostasis has to be maintained for decades without significant loss of functionality. Epithelia such as the oral mucosa achieve this through the self-renewing capabilities of stem cells. These life-long residents of the oral mucosa maintain homeostasis of the epithelium and at the same time have to be able to protect themselves from the main threats to longevity, i.e., stem cell exhaustion and malignant conversion and carcinogenesis. A key pathway in mediating cell cycle arrest in oral keratinocytes is mediated by TGFB superfamily signaling. There is good evidence implicating TGFB signaling as a tumor suppressor pathway in oral cancer. We have previously shown that disruption of TGFB signaling and cell adhesion can lead to oral squamous cell carcinoma. In vitro using oral keratinocytes, we found that TGFB regulates the stem cell compartment, inducing gene expression of quiescent stem cell markers MECP2, XPC, ITGB1 and PDPN. Using multiplex immunofluorescence, we have stained a tissue microarray containing 28 cases of squamous cell carcinoma, 4 adenocarcinoma, 4 metastatic carcinoma, 6 hyperplasia, and 5 adjacent inflammatory as well as normal adjacent and normal tissues. The antibodies selected were MECP2, XPC, ITGB1 and K15 in combination with the proliferation marker ki67 to determine if TGFB signaling regulates markers of stem cell quiescence. This quiescent gene signature highlighted a basal niche for slow-cycling cells, reserve stem cells. During oral disease progression, marker expression was abandoned in favor of increased proliferation. In vivo, a mouse model in which KrasG12D expression was induced and the Smad4 gene deleted specifically in the adult epithelium showed invasive cancers in the oral cavity. These Smad4-negative tumors express Sox9, a marker of human OSCCs and a TGFB target, which we have also observed to be upregulated in human premalignant lesions. Sox9-positive areas in the mouse tumors are associated with a downregulation of quiescent oral stem cell markers Krt15 and Mecp2, same as we observed in immunostainings of human tissues. In conclusion, TGFB and SMAD4 regulate quiescent stem cells and loss of this pathways allows invasive cancers to develop in the setting of oncogene expression. Furthermore, we could show that MECP2 in combination with other markers identifies quiescent basal cells in the human oral mucosa and is lost as TGFB signaling is disrupted in tumorigenesis. Citation Format: Claudia D. Andl, Anna L. Means, James S. Lewis, Alexandra Rothaus, Thomas Andl. TGFB signaling disruption in the formation of oral pre-neoplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4682.