Targeting sphingolipid metabolism in large granular lymphocyte leukemia

Open Access
- Author:
- Leblanc, Francis R
- Graduate Program:
- Molecular Medicine
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- March 02, 2016
- Committee Members:
- "Thomas P Loughran, Jr", Dissertation Advisor/Co-Advisor
Charles H Lang, Committee Chair/Co-Chair
Hong Gang Wang, Committee Member
Edward Joseph Gunther, Special Member - Keywords:
- LGL
large granular lymphocyte
sphingosine kinase
S1P
sphingolipid - Abstract:
- Large granular lymphocyte (LGL) leukemia is a spectrum of rare clonal lymphoproliferative disorders, all of which involve expansion of large granular lymphocytes, either cytotoxic T-lymphocytes (CTL) or natural killer (NK) cells. In normal adults, LGLs represent 10-15% of peripheral blood mononuclear cells (PBMCs) and can be classified into two distinct lineages, as either CD3+ CTLs or CD3- NK cells. Both cells types play important roles in the immune system. LGLs become activated through antigen recognition and undergo significant expansion with subsequent death by apoptosis upon antigen clearage. In LGL leukemia, these LGLs persist. Clinically, LGL leukemia can present with varying disease severity ranging from a slowly progressive indolent and chronic disorder to a highly aggressive acute and fatal leukemia. A high proportion of patients also present with a range of autoimmune conditions, including rheumatoid arthritis. Immunosuppressive regimens using methotrexate, cyclophosphamide or cyclosporine are utilized as front-line treatments in LGL leukemia but are not curative. The aggressive LGL variants have no effective treatment and are refractory to conventional chemotherapy. The lack of effective and targeted therapies results from an incomplete picture of survival mechanisms that contribute to the pathogenesis of leukemic LGLs. In this study, we investigated sphingolipid metabolism and its role in the survival of leukemic LGLs. Sphingolipids are a group of bioactive lipids that maintain cellular integrity and can also mediate signal transduction. The balance between pro-apoptotic sphingolipids, such as ceramide and sphingosine, and pro-survival sphingolipids, such as sphingosine-1-phosphate (S1P), has been shown to regulate cell survival and is known as the “sphingolipid rheostat.” The sphingolipid rheostat is dysregulated in many types of cancer, including LGL leukemia. We identified that the S1P-producing enzymes, sphingosine kinase-1 (SPHK1) and sphingosine kinase-2 (SPHK2), are both upregulated in LGL leukemia patients. We investigated the role of SPHK1 and SPHK2 in leukemic LGL survival and resistance to apoptosis. We show that SPHK1 inhibition with both SPHK1 siRNA and pharmacologic inhibitors (SKI-II, SKI-178) decreases cell viability and induces apoptosis in leukemic LGLs through the targeting of multiple signaling pathways including p38 MAPK, JNK, JAK/STAT and B-cell lymphoma 2 (Bcl-2). Additionally, SPHK2 inhibition with both SPHK2 siRNA and ABC294640, a SPHK2-specific inhibitor, also decreased cell viability and induced apoptosis through the downregulation of myeloid cell leukemia differentiation protein-1 (Mcl-1), an anti-apoptotic Bcl-2 family member. Furthermore, we identify Mcl-1 as an important survival protein in LGL leukemia through direct inhibition with Mcl-1 siRNA and recently developed Mcl-1specific small molecule inhibitors, maritoclax and KS18. Finally, we demonstrate that FTY720 (Fingolimod), a potent immunomodulatory agent, induces apoptosis in leukemic LGLs through the downregulation of acid ceramidase (AC, ASAH1), an enzyme that metabolizes ceramide into sphingosine. We further show that antitumor effects of FTY720 are dependent on autophagy. Taken together, we show that the sphingolipid metabolizing enzymes, SPHK1, SPHK2 and AC, are all important for leukemic LGL survival and are novel potential therapeutic targets. Targeting these enzymes restores the sphingolipid balance to a pro-apoptotic state through the increase of ceramide and decrease in S1P. We also demonstrate for the first time that FTY720 downregulates AC in LGL leukemia. Collectively, these data support SPHK1, SPHK2 and AC as putative targets to develop novel targeted therapies for the incurable disease LGL leukemia.