PROJECT SUMMARY When acute lymphoblastic leukemia (ALL) cells evade the toxicity of cytoreductive chemotherapy, minimal residual disease (MRD) and relapse ensue. MRD remains a major obstacle, which is unaddressed by current therapies. Recently, integrin alpha6 (Itga6) was identified as a novel flow cytometric MRD marker. Why Itga6 marks MRD ALL cells is unknown. We developed novel models to study the emergence of MRD. This novel MRD ALL model enables us to perform in situ characterization of bone marrow (BM) cells neighboring MRD ALL cells and in vivo visualization of interactions of such MRD ALL cells with BM niches in real-time allowing us to study real-time interactions of MRD ALL cells with the BM. Our preliminary data identify Itga6 as a cell surface adhesion receptor for laminin on specific ALL cell subtypes which protects these cells against drug treatment. However, our data also show that Itga6 has a pro-survival function independent of laminin. Moreover, Cre-mediated deletion of Itga6 in murine pre-B ALL cells in vitro reproducibly induces apoptosis. In xenograft model of primary ALL, combination chemotherapy treatment with P5G10, an Itga6-specific Ab, enhanced survival of leukemia bearing mice. As our data show that the functions of Itga6 and another adhesion molecule, Itga4, differ which also is expressed on ALL cells, differ, we will test a novel concept of dual integrin inhibition as treatment strategy for ALL. Preliminary mass spectrometry analysis provides mechanistic correlates of observed Itga6-associated apoptosis which provide the basis for studying the underlying mechanism of how Itga6 promotes survival of ALL cells. Our overall hypothesis and premise for these studies is that MRD pre-B ALL cells are located in a specific niche in the BM, and that Itga6 expression in ALL cells is a critical component that imparts leukemia-initiating cell characteristics, including drug insensitivity, to MRD cells. The following aims will test this hypothesis: Aim 1 will characterize the BM niche cells in contact with MRD ALL cells in situ and in real-time in vivo and in vitro. Aim 2 determines the underlying mechanism of Itga6-promoted MRD ALL survival. Finally, Aim 3 will preclinically evaluate the concept of Itga6 inhibition alone and in combination with Itga4 inhibition to eradicate MRD ALL. Our proposed studies may further establish the proof-of-principle in which interruption of the Itga6/BM interaction abrogates protection of the MRD niche to ALL, changing current concepts of treating ALL to include strategies targeting the MRD niche.