Date of Graduation
12-2013
Document Type
Dissertation (PhD)
Program Affiliation
Experimental Therapeutics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Varsha Gandhi, Ph.D.
Committee Member
Lynne Abruzzo, M.D., Ph.D.
Committee Member
Hesham Amin, M.D.
Committee Member
Jan Burger, M.D., Ph.D.
Committee Member
George Calin, M.D., Ph.D.
Abstract
Chronic lymphocytic leukemia (CLL) is a B-cell malignancy. High levels of Bcl-2 and IAP family proteins are responsible for apoptotic-resistance and accumulation of mature CLL lymphocytes in bone-marrow, lymph nodes and peripheral blood. Besides pro-survival proteins, supporting stromal cells as well as soluble factors in the microenvironment of bone-marrow and lymph nodes provide survival advantage to CLL leukemic cells.
Though the stromal – leukemia cell interactions has been studied extensively, in-depth-knowledge on the regulation of apoptotic pathway proteins in the context of microenvironment is still limited. To address this, the first part of our study focused on comprehensive analysis of 93 gene transcripts from seven families that are key regulators of apoptosis such as Bcl-2, IAPs, NF-kB, Caspase, TNF receptor superfamily (TNFRSF), Death Domain (DED) and Caspase Activation and Recruitment Domain (CARD) families using “real-time PCR apoptosis array-card”. While shorter incubations with supporting stromal cells induced significant changes in 22/93 transcripts including Bcl-2 (6/22), TNFRSF (5/22), DED (5/22), Caspase (2/22) family members, longer incubations induced significant changes in 8/93 transcripts from NF-kB (4/8) and Bcl-2 (2/8) family members. At the protein level, decrease or stable expressions in pro-apoptotic proteins, but a significant increase in anti-apoptotic proteins was observed.
The second portion of this study involved therapeutic manipulation of executioner caspases in restoring apoptotic pathway that is frequently blocked by upstream anti-apoptotic Bcl-2 family members. A novel therapeutic approach that can potentially bypass the functional pro-survival proteins and directly activate terminal executioner procaspases to induce apoptosis was investigated. Executioner procaspase activities are blocked by zinc and zinc removal releases this inhibition. CLL primary cells express high levels of executioner procaspases-7 and -3 compared to normal lymphocytes providing a basis for therapeutic selectivity. Our studies with L14R8, a second generation PAC-1 analog [Procaspase activating compound-1 (PAC-1; Vanquish Oncology)] revealed that L14R8-induced cell death was specific to CLL cells and was independent of prognostic markers and microenvironmental factors. Mechanistically, L14R8 acted through direct activation of executioner procaspases -3 and -7 by removing labile Zinc ions. In summary, our investigations demonstrate cytotoxic actions of L14R8 and elucidate utility of this novel approach for combating CLL.
Keywords
Chronic lymphocytic leukemia, apoptosis pathway, stroma microenvironment, L14R8, B-PAC-1, executioner caspases, therapeutic activation