PROJECT SUMMARY: Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) and their function is regulated by their interactions with the BM microenvironment. Leukemia stem cells (LSCs) have been shown to be capable of initiating and maintaining acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Furthermore, the interactions of LSCs with their microenvironment have been shown to play a critical role in the resistance of LSCs to therapy. The traditional approach of investigating LSCs and anti- leukemic agents’ effects on them has been the 2D monolayer cell culture. As complex cell-matrix interactions continue to be elucidated, it is clear that this model is no longer a sufficient representative of the intricacies of the BM niche. Thus, the development of a model that better mimics this microenvironment is imperative, both to study LSC properties, as well as for testing drug effects in a system that reflects the contributions of the microenvironment. We hypothesize that a culture system capable of recapitulating the BM niche conditions to maintain LSCs will allow for the better understanding of their biology and the discovery of novel therapeutic approaches that can act on LSCs in the BM. We propose to implement novel approaches to evaluate BM-LSC interactions to improve the assessment of the function, drug resistance and stem cell properties of LSCs in a patient-specific manner. Our preliminary data suggest that the 3D spheroids (organoid-like structures) can recapitulate BM niche features, such as hypoxia. In addition, we have observed that the LSCs are better maintained and retain functional stemness properties using the 3D stromal approach compared to more conventional culture systems. Thus, we propose to evaluate AML (with a well-characterized mutational profile) to determine their survival dependencies in the context of niche interactions. The ultimate goal of this project is to identify new therapeutic approaches to target LSCs either directly or through disruption of the BM-LSC protective interactions.