Abstract B33: Toll-like receptor ligands delay acute lymphoblastic leukemia onset via depletion of pre-leukemic cells
Authors: Fidanza Mario , Jo Sumin , Bashir Arnawaz , Grupp Stephan A. , Seif Alix E. , Reid Gregor S. 
Published: May 2015

Abstract
Onset of pediatric acute lymphoblastic leukemia (ALL) depends on the long-term survival and evolution of a pre-leukemic cell population that first arises during fetal development. The detection of chromosomal translocations indicates that about 1% of all newborns harbor early-occurring abnormal cells; however, only 1 in 100 of these infants will progress to leukemia. This reveals that progression to leukemia is not the inevitable fate of these early-occurring abnormal cells.
Infection has long been postulated to play a role in the development and progression of pediatric ALL. While epidemiologic studies have implicated exposure to infection as a protective factor, the impact of immune modulation during the pre-leukemic phase has not been reported. In this study, we use the Emu-RET transgenic mouse, which develops fetal-derived B cell precursor (BCP) leukemia, to investigate whether exposure to danger signals associated with infection influences disease progression via changes in pre-leukemic cell survival.
Using splenocyte cultures established from pre-leukemic mice, we observed significant depletion of the characteristic pre-leukemic abnormal cells following incubation with a panel of ligands for infection-related pattern recognition receptors. The strongest activity was observed with ligands for Toll-like receptors (TLR) 2 (Pam3), 7/8 (R848), and 9 (CpG), with &gt;75% reduction in pre-leukemic cell number (p&lt;0.01). While IFN-gamma made a contribution to pre-leukemic cell depletion achieved with each of these ligands, the use of IFN-gamma deficient mice revealed that it is absolutely necessary for a significant reduction only with R848 (88% depletion with wild-type, 19% with IFN-gamma deficient cells, p&lt;0.05).
To investigate the contribution of direct and indirect pathways to pre-leukemic cell depletion, we purified abnormal cells from pre-leukemic mouse spleens and cultured these cells in the presence or absence of TLR ligands. To date, significant direct cytotoxicity has been observed only with Pam3 (86% reduction, p = 0.0013), while R848 and CpG induce modest direct cytotoxicity. Importantly, however, pre-leukemic cell killing by R848 and CpG was enhanced by the presence of bone marrow from Nod-SCID/gamma common chain-deficient mice, indicating a significant contribution of indirect, lymphocyte-independent killing to the overall depletion of pre-leukemic cells (p&lt;0.0001).
Consistent with these in vitro results, we have previously shown that administration of CpG to leukemia-prone mice early in life reduced the size of the pre-leukemic cell population and significantly delayed onset of disease (p&lt;0.0001). While no mice in the control group survived beyond 250 days of age, several CpG-treated mice remained disease free at one year of age and showed no sign of an expanded BCP population at sacrifice. The contribution of IFN-γ to in vivo depletion of pre-leukemic cells following CpG immune stimulation matched that observed in vitro.
Our results are the first to deomnstrate that exposure to infection-related danger signals alters leukemia progression through the reduction in pre-leukemic cell viability. These findings provide mechanistic support for the protective effects of early-life infections and suggest novel strategies to eliminate the early-occurring abnormal cells that can give rise to initial disease and relapse.
Citation Format: Mario Fidanza, Sumin Jo, Arnawaz Bashir, Stephan A. Grupp, Alix E. Seif, Gregor S. Reid. Toll-like receptor ligands delay acute lymphoblastic leukemia onset via depletion of pre-leukemic cells. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B33.