Organoid culture of acute myeloid leukemia within a native bone marrow microenvironment - PROJECT SUMMARY Our overall goal is to develop a human bone marrow organoid culture method that sustains both normal and leukemic hematopoiesis. A historic inability to culture hematopoietic stem cells (HSC), progenitors, and leukemic blasts beyond days to weeks has hindered fundamental investigations and therapeutic development for hematopoietic pathologies. Notably, a complex bone marrow microenvironment vitally sustains HSC and their leukemic derivatives, containing diverse endothelium, mesenchymal stem cells and osteoid, and providing an essential niche during homeostasis and neoplasia. Conceivably, prior culture systems have been limited by insufficient recapitulation of the structurally and cellularly complex bone marrow microenvironmental niche. In response, we developed an air-liquid interface (ALI) primary bone marrow organoid (BMO) system for acute myeloid leukemia (AML) that propagates intact fragments of bone marrow, preserving osteoid, vascular, and mesenchymal niche components in native spatial orientations. When generated from normal bone marrow, BMO maintain hematopoietic stem cells (HSC) and expand progenitors for >60 days (longest examined), support erythroid, myeloid (including neutrophils) and megakaryocyte differentiation, while BMO transplantation rescues lethally irradiated mice. When applied to leukemia, ALI BMO allow ex vivo expansion of human AML blasts from intact fragments of PDX or primary human bone marrow biopsies, preserving histology, surface markers and mutations for >60 days (longest examined), and undergo successful in vivo transplantation. Our proposal responds to PAR-22-242: Bioengineering Research Grants (BRG), that requests “promising tools, methods and techniques”, “design-directed research” and “organoids that closely mimic physiological conditions”. To exploit this broadly enabling leukemia organoid method, Aim 1 conducts BMO-based live imaging of the bone marrow microenvironment in AML BMO during homeostasis and drug treatment to obtain spatially and time-resolved insights into interactions between AML blasts and the bone marrow microenvironment and modulation by therapies. Aim 2 pursues AML BMO as a tool to define and functionally test AML microenvironmental paracrine signals that are evaluated by pharmacologic blockade for effects on leukemic blasts. Lastly, Aim 3 develops human leukemic BMO as a drug evaluation platform, creating human AML BMO from transplanted PDX models and patient core biopsies, and multiplexing for drug testing, which is correlated to therapy effects on conventional short-term cultures, PDX and prospective clinical outcomes. Importantly, Aim 3 develops methods to enable non-expert institutions to receive live or cryopreserved AML PDO, and to create these cultures themselves. We pursue these goals via a synergistic team of the Multi-PIs Calvin Kuo and Kathy Sakamoto, and Co-investigator Ravi Majeti, towards developing a primary human AML organoid method that leverages a fully native bone marrow microenvironment, with application to studies of leukemia pathogenesis, therapy and precision medicine, and broad potential extension to hematologic diseases in general.
