ABSTRACT Despite improvements in treatment, clinical outcomes for late stage breast cancer (BC) remain poor and there is a critical need for new therapies for metastatic disease. Hyperactivation of receptor tyrosine kinase (RTK) signaling in BC is associated with poor prognosis yet direct RTK inhibitors have had mixed results in clinical trials. RTK signaling drives anoikis resistance, a key metastasis-enabling biology. Thus, defining the underlying mechanisms of this process could identify new druggable targets to treat metastasis. Sphingolipids (SL) are a family of bioactive lipids, classically implicated in cell death, and whose metabolism is altered in cancer. Ongoing studies from the PI’s group, using HER2 amplification as a model system of RTK signaling have identified the SL enzyme dihydroceramide desaturase 1 (DES1) as a key HER2-regulated node in the SL network that was required for anoikis resistance of HER2+ BC cells. In clinical data, high DES1 levels were associated with worse outcomes in HER2+ BC. Moreover, increasing DES1 levels was sufficient to drive in vitro tumorigenicity of HER2+ BC cells whereas DES1 KO reduced in vivo metastasis. Taken together, these novel data have led us to the central hypothesis that DES1 is necessary and sufficient to promote RTK- driven anoikis resistance and metastasis which will be tested by pursuing three specific aims. The first aim will define the role of DES1 in RTK-driven anoikis resistance in vitro and in vivo using Crispr/Cas9 technology to knockout DES1 in RTK-driven BC cells and defining biological effects on in vitro tumorigenicity and in vivo cell survival in the circulation. The second aim will define the mechanism(s) by which RTKs regulate and maintains DES1 activity following ECM detachment using gain of function and loss of function approaches to directly connect DES1 with oncogenic activation of PI3K signaling and the pentose phosphate pathway. The third aim will establish DES1 as a driver of in vivo metastasis and chemoresistance in BC harboring RTK hyperactivation using gain of function approaches to show that increased DES1 is sufficient to promote an aggressive phenotype in RTK-activated BC and can mediate resistance to the targeted HER2 therapy lapatinib. Collectively, these studies will establish DES1 as a key effector of oncogenic RTK signaling and as a novel druggable target, effective at overcoming anoikis resistance and useful for treatment of metastatic BC.