The Pennsylvania State University The Graduate School Department of Molecular Medicine ROLE OF SPHINGOLIPID SIGNALING IN PATHOGENESIS OF LARGE GRANULAR LYMPHOCYTE LEUKEMIA A Dissertation in Molecular Medicine by Mithun Vinod Shah © 2009 Mithun Vinod Shah Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2009 ii The dissertation of Mithun Vinod Shah was reviewed and approved* by the following: Thomas P. Loughran, Jr. Professor of Medicine Dissertation Advisor Co-Chair of Committee Rosalyn B. Irby Assistant Professor of Medicine Co-chair of Committee Gary Clawson Professor of Pathology, and Biochemistry and Molecular Biology Edward J. Gunther Assistant Professor of Medicine Charles H. Lang Director, Molecular Medicine Graduate Program *Signatures are on file in the Graduate School iii ABSTRACT Large granular lymphocyte (LGL) leukemia is a disorder of mature cytotoxic cells. LGL leukemia is characterized by accumulation of cytotoxic cells in blood and infiltration in bone marrow and other tissues. Leukemic LGL could arise from expansion of either CD3+ CD8+ T-cells (T-cell LGL leukemia or T-LGL leukemia) or those arising from CD3- natural killer (NK)-cells (NK-cell LGL leukemia or NK-LGL leukemia). LGL leukemia is a rare disorder consisting of less than 5% of non-B cell leukemia. Clinically, LGL leukemia can manifest along a spectrum of disorders ranging from slowly progressing indolent disorder to an aggressive leukemia that could be fatal within months. About fifty percent of LGL leukemia patients also present with variety of autoimmune conditions, rheumatoid arthritis being the most common one. Normally, following antigen clearance, cytotoxic T-lymphocytes (CTL) become sensitive to Fas-mediated apoptosis resulting in activation-induced cell death (AICD). In contrast, the leukemic LGLs are resistance to Fas-mediated apoptosis. It is believed that constitutively active survival signals keep leukemic LGLs alive in the face of functional Fas-apoptotic machinery. Previously, constitutive activation of Jak-Stat signaling pathway, PI3k-Akt pathway, and Ras-Mek-Erk signaling pathways have been described in leukemic LGL. In this study, we identified unique gene signature from PBMC of thirty LGL leukemia patients. Pathway-based analysis of gene signature identified that sphingolipid metabolism and signaling are differentially regulated in leukemic LGL. We also showed that unlike CD8+ cells from healthy donors, sphingosine-1-phosphate (S1P) receptor iv S1P5 (EDG8) is the predominant S1P-receptor (S1PR) leukemic LGL. Disruption of balance between pro- and anti-apoptotic sphingolipids (also known as sphingolipid rheostat) leads to selective induction of apoptosis in leukemic LGL. We further showed that sphingosine-1-phosphate (S1P)-mediated signaling induces resistance to Fas- mediated apoptosis in normal activated PBMC. On the other hand, inhibition of S1P- mediated signaling by FTY720 restored Fas-sensitivity in leukemic LGL. Collectively these results suggest that S1P-S1P5 mediated signaling may contribute to abnormal survival of leukemic LGL that is mediated by Gα12 signaling. We further investigated how FTY720 induced cell death in leukemic LGL. FTY720 induced cell death in leukemic LGL that is insensitive to pertussis toxin (PTX) suggesting a role of Gα12/13-mediated signaling in survival of leukemic LGL. FTY720- mediated cell death is independent of sodium orthovanadate or okadaic acid (OA) suggesting that FTY720 mediates cell death independent of phosphatases. Our results indicate that FTY720-mediated cell death in leukemic LGL was independent of action of extrinsic- or intrinsic caspase cascades suggesting a possibility that FTY720 mediated apoptosis-like programmed cell death rather than classical apoptosis. FTY720 damaged mitochondrial membrane prior to induction of cell death and downregulated MCL1 independent of STAT3 activity in one patient sample but not in others. FTY720 is a novel immunomodulatory agent currently being evaluated for post renal transplant patients and in patients with autoimmune conditions such as multiple sclerosis. Recent work has identified its role as a novel antineoplastic drug as well. Due to significant association of LGL leukemia with autoimmune conditions, it is possible that FTY720 may be of therapeutic benefit to these patients. v TABLE OF CONTENTS LIST OF FIGURES .....................................................................................................ix LIST OF TABLES.......................................................................................................xii ACKNOWLEDGEMENTS.........................................................................................xiii Chapter 1 Clinical features of Large Granular Lymphocytes.....................................1 Clinical features of LGL leukemia ..............................................................................2 Signs and Symptoms.............................................................................................2 Immunophenotype and Diagnosis ........................................................................3 Treatment .....................................................................................................................4 Survival Signaling in LGL Leukemia..........................................................................5 Role of Ras-Mek-Erk Signaling Pathway....................................................................5 Dysregulation of Ras-Mek-Erk signaling in pathogenesis of LGL leukemia ......6 Therapeutic implications ......................................................................................7 Role of PI3k-Akt Signaling Pathway...........................................................................8 Dysregulation of PI3k-Akt pathway in LGL leukemia ........................................9 Therapeutic Implications...............................................................................10 Role of Nuclear factor kappa-B (NF-κB) Signaling Pathway .....................................10 Dysregulated NF-κB signaling in survival of leukemic LGL ..............................11 Role of Jak-Stat Signaling Pathway.............................................................................12 Dysregulated Jak-Stat pathway in pathogenesis of LGL leukemia ......................13 Therapeutic implications ...............................................................................14 References....................................................................................................................16 Chapter 2 Activation-induced Cell Death and its Dysregulation in Leukemic LGL..20 Activation-induced cell death in T-cell homeostasis...................................................20 Uncoupling of AICD in LGL leukemia................................................................22 Biology of Fas-mediated apoptosis ......................................................................22 vi Fas-resistance in LGL leukemia ..................................................................................23 Dysregulation of Fas-mediated apoptosis in leukemic LGL ................................23 Role of soluble Fas and FasL in LGL leukemia...................................................26 Abnormal formation of DISC in leukemic LGL ..................................................27 Materials and Methods.................................................................................................28 Patient consent...............................................................................................28 Cell culture and CD8+ T-cell enrichment .....................................................28 RNA isolation and global gene expression analysis......................................30 EASE analysis ...............................................................................................32 Results ..........................................................................................................................32 Comparison of the gene expression signature of naïve normal cells with activated normal cells....................................................................................32 Comparison of the gene expression signature of naïve normal cells with leukemic LGL................................................................................................34 Comparison of the gene expression signature of activated normal cells with leukemic LGL................................................................................................35 Dysregulated expression of apoptosis related genes in leukemic LGL................37 Apoptosis related genetic signature unique to leukemic LGL..............................40 GenMAPP analysis of apoptosis related genes in leukemic LGL.................41 Validation of microarray analysis................................................................................43 NKp46 ...........................................................................................................43 Serine Proteinase Inhibitor 9 (SERPINB9) ...................................................45 Other genes....................................................................................................47 Discussion....................................................................................................................48 References....................................................................................................................50