Summary Mucoepidermoid carcinomas (MEC) are the most common type of malignant salivary gland tumor (SGT) with advanced MEC often being fatal due to resistance to conventional therapies. Despite their prevalence in salivary malignancies and occurrence in other tissues throughout the body, a thorough understanding of the key molecular events leading to their development has not been fully elucidated. In fact, the incomplete molecular characterization of SGTs was identified by the NIH/NIDCR as a major roadblock to the creation of new clinical diagnostics and therapies. Thus, a comprehensive understanding of the mechanisms driving MEC development and progression is critical to developing better therapeutic interventions. The Aims of this proposal address these significant knowledge gaps through the seamless integration of expertise, tools, and technologies, assembled by our group to investigate how coordinated regulation of two key signaling pathways by a CRTC1-MAML2 (C1/M2) fusion oncogene regulates differentiation and tumor grade in salivary MEC. The C1/M2 fusion is recognized as a key defining feature occurring in over 50% of MEC cases, which reprograms the CREB transcriptional network to drive tumorigenesis. However, in addition to CREB, our recent work and exciting Preliminary Studies now reveal that the C1/M2 oncoprotein also harbors gain-of-function properties that enable MYC binding and co-activation of the MYC transcriptional network. Mechanistically, our data reveal an etiologic role for C1/M2 in the altered cell heterogeneity associated with advanced salivary MEC, identify IGF-1 signaling as a hallmark of fusion positive MEC, and uncover antagonistic roles for C1/M2-CREB versus C1/M2-MYC in promoting differentiation or de-differentiation, respectively. Moreover, we developed and validated the first autochthonous mouse model of salivary MEC that faithfully mimics the genetic and pathologic features of human disease. Therefore, our central hypothesis states that C1/M2 influences intra- tumoral heterogeneity during salivary MEC development and progression by balancing expression of CREB and MYC transcriptional programs that control cell differentiation. To interrogate the molecular and cellular events that initiate, maintain the malignant state, and mediate response to therapy, we also developed an innovative optical reporter and mouse model that permits longitudinal characterization of tumorigenesis. In Specific Aim 1 we will establish whether IGF-1 regulation promotes MEC tumorigenesis and drug resistance. In Specific Aim 2 we will elucidate the mechanisms governing C1/M2 interactions with MYC and the functional significance of these interactions, and in Specific Aim 3 we will determine the clinical significance of regulating CREB versus MYC in relation to MEC pathology.