Summary Therapeutic resistance and tumor relapse present as major barriers to achieving a definitive cure for cancer. This challenge is especially relevant for patients with pancreatic ductal adenocarcinoma (PDAC), who are largely diagnosed at advanced stages and face low survival odds. Recent studies have revealed that tumors are complex ecosystems consisting of coexisting subclonal populations that each harbor a unique genomic landscape. Indeed, tumors are constantly adapting in response to external perturbations such as therapeutics, and clones capable of surviving treatment are evidence of evolved resistance to therapeutics and may provide the foundation for relapse. While the role of mitochondria in tumors has been largely neglected, recent studies have demonstrated that OXPHOS can contribute to treatment resistance as well as several other processes such as invasion and metastatization. Here, we will leverage out our novel Clonal Replica Tumors (CRTs) platform to test the hypothesis that heterogenous mitochondrial activity across different clonal lineages plays a central role in determining tumor response to therapy, and thus contributes to the development of therapeutic resistance and tumor relapse in PDAC. Our CRT platform enables the testing of multiple pharmacological disruptors or other external factors in parallel in animals bearing patient-derived xenotransplanted (PDX) tumors with identical clonal composition. This approach allows us to explore how intra-tumor mitochondria functional diversity is shaped by genomic heterogeneity as well as whether and how this diversity contributes to therapeutic resistance. By focusing on treatment-naïve subclonal lineages with distinct responses to therapy isolated from early passage pancreatic cancer PDXs, we will investigate the following aims: 1) explore the role of genomic heterogeneity in shaping mitochondria functional diversity and define mitochondrial molecular signatures that predict treatment response; 2) elucidate the role of mitochondria in mediating pharmacological resistance; 3) determine the effects of targeting mitochondria on tumor clonal architecture and construct a 3D map of tumor resistance. Ultimately, we will explore the therapeutic benefits of targeting mitochondria to prevent therapeutic resistance and relapse in pancreatic cancer. We are confident that our study is responsive to the NIH/NCI mission to improve patient outcomes, as it addresses fundamental questions about how intratumoral mitochondria heterogeneity and distinct mitochondrial phenotypes influence treatment response to drugs and sustain tumor relapse. We further anticipate that our research will have an immediate translational impact through the identification of new biomarkers that can be used to identify patients who may benefit from the OXPHOS inhibitors currently under clinical investigation.