Therapeutic Potential for Toll-Like Receptor 2 Agonists In Pediatric Acute Lymphoblastic Leukemia (pre-B ALL): Induction of Apoptosis and Anti-ALL Immunogenicity
Authors: Rolf Nina , Kariminia Amina , Ivison Sabine , Schultz Kirk R. 
Published: October 2019

Abstract
Abstract 870

Introduction:
Despite high cure-rates achieved by an intense chemotherapy regimen in pediatric pre-B ALL, 20% of children suffer relapse after which they face limited therapy options and poor prognosis. New treatment options are urgently needed.

Background:
Hematopoietic cell transplantation (HCT) has become an important therapy for ALL-relapse, both by eradicating leukemic blasts through myeloablative chemotherapy and by inducing a graft-versus-leukemia effect (GVL). Inadequate post-HCT immune responses can increase risk of relapse, while a GVL effect often comes at the expense of graft-versus-host disease leading to significant morbidity and mortality. Stimulation of Toll-like receptors (TLRs), which detect conserved pathogen- and stressed-self-derived ligands, represents a possible strategy for augmenting anti-ALL immunogenicity. TLR stimulation of antigen-presenting cells induces the expression of co-stimulatory molecules and cytokines, resulting in Th1-polarized immune responses important for anti-tumor responses. Furthermore, it has been shown that TLR stimulation can increase the sensitivity of hematological malignancies to conventional chemotherapeutics (Spaner et al, Leukemia 2010). We have previously used agonists of endosome-localized TLR9 (synthetic CpG-ODN) to enhance anti-ALL immunity (Reid et al, Blood 2005). This approach, which was successful in eliminating leukemia and extending survival in a syngeneic mouse model of minimal residual disease (Seif et al, Blood 2009), is now entering a phase I clinical trial (TACL group). However, our recent research demonstrated that TLR2 receptors, localized on the surface and thus independent of endocytosis, are more abundantly and consistently expressed on pre-B ALL cells. Unlike most other TLRs, which are functionally active as homodimers, TLR2s can form heterodimers with TLR1 or TLR6 suggesting that TLR2 ligands can modulate multiple downstream signaling pathways. Altogether, TLR2 agonists may have better efficacy in generating anti-B-ALL immunity, apoptosis and chemosensitivity.

Objective:
We tested the hypothesis that i) TLR2 agonists increase pre-B ALL blast sensitivity to doxorubicin (DOX) and asparaginase (ASP) in vitro. We further investigated if ii) distinctive synthetic TLR2 agonists (TLR2/6: Pam2CSK4=Pam2, TLR2/1: Pam3CSK4=Pam3 and FSL-1) differ in their ability to 1) transduce specific signaling pathways, 2) induce apoptosis, and 3) augment pre B-ALL cell immunogenicity.

Methods:
Pre-B ALL cell lines pre-treated with TLR2 agonists were compared to untreated samples in vitro using the following methods: 1) NFkB phosphorylation (pNFkB) and IkB degradation was detected by flow cytometry in a time and dose-dependent manner; 2) Induction of apoptosis/necrosis of blasts by TLR2 stimulation was examined by flow cytometric detection of AnnexinV/7AAD. 3)In vitro augmentation of immunogenicity was investigated by measuring induction of co-stimulatory molecules and increment of allogeneic T-cell proliferation.

Results:
1) Pam2 rapidly and potently induced NFkB signaling (pNFkB 23–42% at 1ug/ml after 15–30min), while Pam3 (10ug/ml) displayed a slow and continuous increment at 60min, thus underlining the differences in signaling kinetics. 2) Pam3 stimulation induced significant, dose-dependent cell death: 36% (6hr), 66% (24hr), 81% (48hr) at 10ug/ml. Unexpectedly, both TLR2/6 agonists, Pam2 and FSL-1, did not induce comparable degree of cell death. Thus, Pam3 killed pre-B ALL cells more potently than therapeutic levels of Doxorubicin (0.005ug/ml) at the earliest time point of 6hr (8.5%) with equal cytotoxicity at 24hr (44%) and 48hr (93%). 3) All TLR2 agonists induced a comparably high expression of CD40/80/86 after 48hr (83-93%/63-76%/84-86%). However, only Pam3 induced a dose-dependent, early CD40 expression at 6hr (32%) and 24hr (60%). Furthermore, blast pre-treatment with Pam3 (but not Pam2) increased the sensitivity to ASP (49%/73% live cells with/without Pam3, respectively). Finally, there was a 30% increase in immunogenicity of pre B-ALL blasts by Pam3 in T-cell alloreactivity studies when compared to medium.

Conclusion:
TLR2 agonists increased anti-ALL immunogenicity in vitro. Pam3 also had strong cell-death inducing qualities and sensitized pre-B ALL blasts to chemotherapy. This supports that TLR2 agonists have promise for improving relapsed pre-B ALL cure-rates.

Disclosures:
No relevant conflicts of interest to declare.