PROJECT SUMMARY/ABSTRACT With 5-year survival rates of 10-15%, metastatic renal cell carcinoma (RCC) is largely incurable. Tyrosine kinase inhibitors (TKIs) are the largest and most widely used class of drugs for RCC treatment (6 FDA-approved drugs), but resistance routinely develops. The first TKIs were approved by the FDA nearly 2 decades ago, but how resistance arises has remained a mystery. RCC TKIs were developed to target vascular endothelial growth factor receptor 2 (VEGFR2), which plays a critical role in angiogenesis and RCC biology. However, a role for VEGFR2 in mediating the anti-tumor response remains to be demonstrated, and most RCC TKIs target multiple other kinases. Understanding resistance mechanisms has been instrumental in dissecting how oncology drugs precisely work. As such, how RCC TKIs function remains enigmatic and this represents a significant, longstanding, and critical knowledge gap. Furthermore, understanding how kinase inhibitors exert their anti- tumor effect and how resistance develops often yields new avenues for drug development. Indeed, relevant targets for other kinase inhibitors have been identified and validated across multiple tumor types, which has enabled the development of second- and third-generation inhibitors. This proposal seeks to understand how cabozantinib, arguably the most potent FDA-approved TKI for RCC, functions to inhibit tumor growth and how resistance develops. Cabozantinib inhibits a family of tumor-driving kinases with roles in tumor cells (such as AXL, MET, RET and KIT) and the tumor microenvironment (such as VEGFR2). One potential explanation for why it remains to be determined how RCC TKIs function is that inhibition of kinases in the tumor microenvironment (TME) plays a critical role in their anti-tumor effects. This proposal will evaluate not only how cabozantinib affects tumor cells, but also how it affects the TME. However, dissecting the role of the TME in the anti-tumor response is challenging and innovative approaches are needed. The proposal will leverage a pioneering mouse model to dissect, for the first time, the role of the TME in mediating cabozantinib anti-tumor response. If successful, these studies will establish how cabozantinib exerts its anti-tumor activity. By defining how cabozantinib functions and how resistance is acquired, we will be poised to develop strategies to overcome resistance. The innovative paradigm presented herein has broad application to dissecting the role of the microenvironment in mediating the action of any cancer drug, and the ensuing discoveries may pave the way for the next generation of inhibitors and enable rational combinations.