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Modeling Central Nervous System Involvement in Acute Lymphoblastic Leukemia Stephen M. Akers Infiltration of the central nervous system (CNS) by leukemic blasts represents one of the problematic disease manifestations of acute lymphoblastic leukemia (ALL). Modern prophylactic measures have decreased the rate of CNS involvement in ALL. However, they produce adverse side effects, including cognitive dysfunction, seizures, and growth retardation, which have unique implications in pediatric patients that constitute the bulk of ALL cases. While there has been significant research into how ALL cells are nurtured in sanctuary sites, such as the bone marrow, there is a paucity of literature reporting on the mechanisms through which ALL cells migrate into the CNS and how they interact with cellular constituents of the CNS to evade treatment in this unique sanctuary site. To this end, the overall goals of the current body of work were to understand how ALL cells interact with human brain-derived microvascular endothelial cells (HBMEnd) and to understand how cellular constituents of the subarachnoid space of the CNS alter ALL cell response to chemotherapeutics routinely used in the prophylaxis of CNS leukemia. Using in vitro models, we investigated the interaction between ALL cells and HBMEnd to understand the functional significance of coincident VE-cadherin and PECAM-1 expression by ALL. Based on our observation that induction of adhesion molecules that are typically increased subsequent to inflammation did not occur following interaction of ALL cells with endothelial cells, we explored adhesion molecules expressed constitutively by ALL cells that could enhance leukemic cell adhesion to HBMEnd. Evaluation of primary ALL samples, including leukemic cells isolated from CSF, demonstrated that VE-cadherin and PECAM-1 are co-expressed on the tumor cell surface. Based on the classical role of VE-cadherin and PECAM-1 mediating homotypic interactions between adjacent endothelial cells, we hypothesized that expression of these two proteins by ALL cells would enhance their interaction with HBMEnd. Using lentiviral-mediated expression of these two proteins and neutralization of protein function with specific antibodies, we demonstrated expression of VE-cadherin and PECAM-1 by ALL enhanced the adhesion of ALL to HBMEnd, while expression of PECAM-1 enhanced ALL adhesion to, and migration through, HBMEnd. We also investigated the contribution of astrocytes, choroid plexus epithelial cells, and meningeal cells to alterations in leukemic cell survival during treatment with chemotherapeutics routinely used for the prophylaxis of CNS involvement in ALL. As these cells from the CNS have been documented to express soluble factors and adhesion molecules similar to cells resident in the bone marrow that enhance the survival of ALL cells following chemotherapy treatment, we hypothesized that culture of ALL cells with cellular constituents of the subarachnoid space would promote ALL survival following exposure to cytarabine, dexamethasone, and methotrexate. We demonstrated that ALL cells migrate towards chemotactic stimuli secreted by astrocytes, choroid plexus epithelial cells, and meningeal cells. Additionally, we documented the physical interaction of ALL cells with these three CNS-derived cell types. Finally, through the use of in vitro co-culture models, we showed that meningeal cells, choroid plexus epithelial cells, and astrocytes confer protection to ALL cells from chemotherapy-induced cell death using drugs typically found in CNS prophylactic regimens. The research described herein provides foundations for understanding how ALL cells interact with endothelial cells and cells of the subarachnoid space that would be important for invasion and survival in the CNS, respectively. Furthermore, these studies serve as a springboard for further investigations into the mechanism used by ALL cells to infiltrate the CNS as well as investigations to elucidate the exact soluble factors and adhesion-mediated signaling events that enhance ALL survival in the CNS. Ultimately, this work may improve our understanding of CNS involvement in ALL and may allow for the development of strategies to prevent CNS leukemia and minimize the need for treatment in this sensitive anatomical site where treatment-induced toxicity is of significant concern. ACKNOWLEDGEMENTS The pursuit of this degree over the past four years has been the most challenging academic endeavor I have undertaken. Without the support and guidance from professional colleagues, friends, and family, the completion of this work would not have been possible. For all of your help, I am truly grateful. I must first thank my mentor Dr. Laura Gibson. Under her guidance, I have matured from a second year medical school student barely knowing the techniques involved in laboratory investigations to a doctoral candidate capable of critically analyzing a problem and designing investigations to solve them. Through her example, she has taught me how to become organized, how to make a plan, and how to work with efficiency in all I do. These traits, which I lacked before (and amazingly still made it to this point academically) and continue to improve upon, will be lifelong lessons that will serve me in every future endeavor. I want to also thank Dr. Gibson for her seemingly eternal optimism over the course of these projects. When I had doubts about the completion of this work, she remained confident. I would also like to thank those who have contributed to the completion of the studies presented in this dissertation. This includes all of the authors: Dr. Heather O’Leary, Dr. Fred Minnear, Dr. Michael Craig, Dr. Jeff Vos, Dr. Jim Coad, Stephanie Rellick, and Jim Fortney. Additionally, those who have helped with core facilities, molecular techniques, patient samples, and other materials Dr. Kathleen Brundage, Dr. Karen Martin, Dr. Alexey Ivanov, Kim HuffmanJones, Deirdre Bulian, and Dr. Gerry Higa. Also, those whose intellectual input has helped guide this work includes my committee members, Dr. Scott Weed, Dr. Jame Abraham, Dr. Jun Liu, Dr. Fred Minnear, and Dr. Linda Vona-Davis. While the work that is presented herein is a direct result of the contributions of these professional colleagues, it is the result of a lifetime of support of my friends and family that has brought me to this point. I must first start with expressing my gratitude to all of the past and present members of the Gibson lab. To Audrey, Debbie, Heather, Jim, JZ, Sree, and Steph, I

Modeling central nervous system involvement in acute lymphoblastic leukemia