This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT- CA-21-009. This supplemental proposal seeks to uncover the mechanism of synergy and develop an optimized regimen of the combination of a microtubule-disrupting agent, eribulin, and a PI3K-inhibitor in PDX models of breast cancer. The work will be conducted as a national collaboration between Washington University in St. Louis and BIDMC/Harvard Medical School in Boston. In her pre-clinical work, Dr. Ma at Washington University in St. Louis has shown that the combination of eribulin and the PI3K-inhibitor copanlisib greatly extends progression-free survival in eight PDX models of TNBC. This novel concept is now being carried forward into a clinical trial in patients with metastatic TNBC (NCT04345913). Her discovery was surprising as PI3K-inhibitor benefit so far had been restricted to ER+PIK3CAmt breast cancer. The exact mechanism and, based on the mechanism, best timing of eribulin and PI3K-inhibitor will be determined in the proposed work. Our Parent Proposal is slated to develop PI3K-inhibitor (PI3Ki) combinations for patients with PIK3CA- mutant breast cancer. PI3Kinase is a nodal point of the intracellular signaling machinery that drives cell division of breast cancer cells. PIK3CA-mutant breast cancer can be targeted with alpelisib, a recent FDA-approved PI3K-a inhibitor now widely used in the metastatic setting in conjunction with estrogen receptor blockade. Our Supplemental Proposal is an extension of Aim 2, To determine in vivo if the metabolic changes induced by PI3K- inhibition are predictive of cancer treatment responses and of Aim 3, To determine in vivo if time-staggered PI3K- inhibition can enhance the efficacy of antineoplastic treatments for endocrine-resistant PIK3CA-mutant BC of our Parent Proposal. In this project, we hypothesize that PI3K-inhibition is a metabolic intervention that, if applied strategically following microtubule disruption, can deepen and prolong remissions obtained with microtubule- disrupting drugs, which are widely used to treat metastatic breast cancer. We will employ in vitro imaging and metabolomic studies and in vivo imaging with 18FDG-glucose and 13C-pyruvate to deep-probe glycolysis in response to chemotherapy, PI3K-inhibition and their combination and test if these imaging modalities can predict responses. The team at WashU (Dr. Cynthia Ma, medical oncology and preclinical mouse work, Dr. Kooresh Shogi (quantitative PET-imaging, Dr. Cornelius von Morze (quantitative MRI imaging) and at BIDMC/Boston (Dr. Gerburg Wulf, pre-clinical mechanistic studies and Dr. Aaron Grant, pioneer in 13C-pyruvate imaging) have established a MTA for transfer of the PDX models and will conference bi-monthly to make this supplemental project happen within a year.