Abstract: In early breast carcinogenesis, neoplastic cells grow in multiple layers towards the lumens of ducts, which subjects the periluminal cells to harsh conditions of low oxygen, low pH, and nutrient deprivation. Adaptation to these harsh conditions is a pre-requisite for survival of incipient tumor cells. Adaptations are initially acute and reversible, but eventually Darwinian selection results in cells with hardwired phenotypes. A prominent example of this is aerobic glycolysis, or the Warburg Effect (WE), wherein cells are hard-wired to ferment glucose, even in the presence of oxygen. Notably, a WE is highly correlated with a cancer’s metastatic potential and poor outcome. Hence, a major question in carcinogenesis is: “What are the mechanisms by which a harsh microenvironment eventually selects for hard-wired (heritable) phenotypes, such as a WE?”. Rather than simply selection of pre-existing phenotypes, we contend that the microenvironment actively induces phenotypic diversity through a systematic set of epigenetic and genetic alterations. To address this question, we combine preliminary data from three different approaches that are all focused on the eco-evolutionary dynamics occurring during carcinogenesis: In the first, we have subjected benign breast cancer and epithelial cells to harsh conditions encountered in DCIS and have observed that the cells that survive these selections exhibit a WE. We selected three clones and applied single cell RNA sequencing and single cell ATAQ sequencing as well as whole exome sequencing to map the transcriptome, epigenome, and mutation patterns of the selected clones compared to their parental normal cells. This will form the model system to be analyzed throughout the current proposal. In the second line of investigation, we have documented the profound epigenetic changes that occur during progression of multiple myeloma (MM) from pre-malignant to metabolically active disease. We hypothesize that these observations in MM can provide a framework to predict and interpret the changes that breast cancer cells undergo as they transition from a benign non-glycolytic to an aggressive glycolytic state. In the third line of investigation, we have outlined a continuum starting with epigenetic changes and show how these result in permanent mutational or chromosomal changes. This latter work provides a framework with which to predict and interpret how microenvironment-induced epigenetic changes can eventually lead to hardwired genetic changes that are observed in aggressive glycolytic breast cancer. By combining these approaches, we propose to decipher the mechanisms whereby microenvironmental stress-induced genome evolution results in hard-wired phenotypic adaptations, represented by a WE. At the end this study, we expect to have a more complete and comprehensive understanding of the environmentally-induced epigenetic and genetic changes that occur during carcinogenesis, and how these relate to hard-wired phe...