Abstract A81: Early TLR-mediated killing of leukemia in bone marrow is correlated with durable protection against B cell precursor acute lymphoblastic leukemia
Authors: Jo Sumin , Elzen Peter van den , Reid Gregor S.D 
Published: February 2019

Abstract
The ability of Toll-like receptor (TLR)-induced innate immune responses to activate adaptive immune responses offers considerable therapeutic potential for cancer treatment, most likely as part of combination therapies. The potent immunostimulatory capacity of TLR agonists provides means to generate antigen-specific antitumor T cell responses from antigen-nonspecific innate immune stimulation.
B cell precursor (BCP) acute lymphoblastic leukemia (ALL) exhibits the very low mutational burden common to other pediatric cancers. Despite this limited neo-antigenicity, we previously reported the ability of CpG oligodioxynucleotides (ODN), a ligand for TLR9, to mediate anti-leukemia activities that establish long-term protection against ALL outgrowth in a transplantable syngeneic model using cell lines. CpG ODN also reduced the burden of primary human ALL in mouse xenografts, primarily via induction of NK cell-mediated cytotoxicity. However, our recent demonstration that endosomal TLR agonists generated distinct effects on pre-leukemic BCP cells, coupled with the divergent effects of TLR ligands on the immunogenicity of primary human ALL cells suggest that there are significant variables that contribute to the overall outcome of TLR stimulation in the context of BCP cell malignancy. In this study, we compared the ligands for TLR3 (polyI:C), TLR7/8(R848) and TLR9 (CpG ODN) for their ability to elicit control of primary ALL cells and correlated protection with early innate immune activation to identify the necessary component of a long-term protective anti-leukemic response.
We adoptively transferred syngeneic primary ALL cells to wild-type mice then administered TLR ligands starting at day 7 for 3 doses every 4 days. 21 days after leukemia challenge, we evaluated the disease burden in the peripheral blood, spleen, and bone marrow of recipients. While a significantly lower disease burden was observed in all three anatomical compartments of mice treated with polyI:C, CpG ODN- and R848-treated mice failed to control the early outgrowth of transferred cells in the spleen and bone marrow, respectively. While R848-treated mice had a slightly delayed disease progression with median survival (MS) of 40 days compared to that of 28 days of PBS-treated control group, CpG ODN treatment conferred a significant survival advantage where over 57% of treated mice achieved a prolonged disease-free status. Despite successfully inducing systemic anti-ALL responses, poly I:C treatment failed to confer durable protection, achieving only a modest increase in disease-free survival in treated mice (MS=42 days). Moreover, CpG ODN-induced long-term survival in the absence of effective early immune-mediated control of ALL in spleen implies that bone marrow is not only an important homing niche supporting the survival and proliferation of leukemia blasts prior to migration into the circulation, but a critical target site for controlling disease progression.
Furthermore, to investigate the early innate immune responses associated with the differential overall effects induced by TLR agonists, we evaluated the activation status of innate immune system using RAG-knockout mice. A single administration of CpG ODN, but not polyI:C nor R848, was sufficient to rapidly induce anti-ALL immune responses involving activated natural killer and antigen-presenting cells in the bone marrow of the mice bearing ALL in 4 days. This suggests that CpG ODN has superior capacity to initiate innate immune-mediated anti-ALL immune responses and activate subsequent antigen-specific T cell responses.
TLR-mediate innate immune responses induced early in life are distinct from that induced in adult life. Given the peak incidence of pediatric ALL between 3-5 years of age, we are currently examining the influence of age on the TLR-induced anti-ALL immune responses to further evaluate the therapeutic benefits of TLR ligands.
Our results demonstrate the differential magnitude of tissue-specific innate immune-mediated control of leukemia outgrowth induced by TLR stimulations correlating with durable disease-free survival. These findings provide mechanistic explanation for TLR-mediated protective immune responses and highlight the potential target site necessary for effective elimination of leukemia cells.
Citation Format: Sumin Jo, Peter van den Elzen, Gregor S.D Reid. Early TLR-mediated killing of leukemia in bone marrow is correlated with durable protection against B cell precursor acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A81.