Toll-Like Receptor Agonists Induce Immunogeneicity and Apoptosis of Acute Myeloid Leukemia Cells.

Abstract

Acute myeloid leukemia (AML) is a common form of acute leukemia and remains a difficult disease with poor survival in patients who have failed standard therapy. New therapeutic strategies are needed to achieve longer survival and improve cure rates in AML patients. Toll-like receptor (TLR) agonists have been shown to elicit anti-leukemia effects in murine AML models. However, TLR expression profile of human AML cells is unknown. We analyzed TLR1-10 mRNA expression in purified AML cells from 41 patients with different AML subtypes (M0, M1, M2, M3, M4, or M5; n > 5 per group) by real-time RT-PCR. The majority of AML samples expressed high level of TLR2, 4, 7, 8, low level of TLR1, 5, 9, 10, and undetectable level of TLR3. Significant higher TLR4 and TLR7 expressions were detected on M4 and M5 subtypes of AML cells. Triggering TLR4 or TLR7 with specific TLR agonists (Monophosphoryl Lipid A or Imiquimod) significantly increased the surface expression of molecules essential for T cell activation (CD54, CD80, CD86) on AML M4/M5 cells and enhanced T-cell mediated proliferative responses against AML cells. Thus, TLR signaling enhances the immunogenicity of AML M4/M5 cells and makes them more suitable targets for T cell mediated attack. Most importantly, TLR7 agonist strongly induced apoptotic death of primary AML M4/M5 cells and inhibited the growth of TLR7-expressing AML cell lines (U937, HL-60, KG-1) in culture in a drug dose dependent manner. The addition of TLR7 agonist at 10 ug/ml fully induced apoptosis of AML cells and inhibited the growth of AML cell lines, as confirmed by viable cell counts and TMRE staining. Intracellular staining demonstrated that TLR7 agonist treatment significantly down-regulated the signal transducer and activator of transcription (STAT)3 and STAT5 protein expression in AML cells. These results suggest that TLR7 agonist-induced apoptosis of AML cells is likely via inhibition of STAT3 and/or STAT5 signaling pathway. To study the in vivo effects of TLR7 agonist against human AML cells, primary AML M4/M5 cells or a monocytic AML cell line (U937) were injected i.p. into NOD-SCID IL2Rgamma<null> mice with or without subsequent TLR7 agonist treatment. Mice receiving TLR7 agonist treatment (1 mg/kg daily i.p. infusion for 5 days) significantly reduced tumor burden with substantially lower numbers of engrafted leukemia cells detected in these xenograft mice. Flow cytometry results confirmed that residual AML cells recovered from mice treated with TLR7 agonist were apoptotic with down-regulated expression of TMRE and STAT3/STAT5, confirming previous in vitro findings that TLR7 agonist-treated AML cells are programmed to die. The antitumor efficacy of systemic administration of TLR7 agonist in NOD/SCID mice with established human AML is being investigated using these xenograft mouse models. In summary, our results provide the first report of TLR expression profile of human AML cells and demonstrate that TLR targeting of AML cells can increase the immunogeneicity of leukemia cells and directly induce AML apoptosis in vitro and in vivo, providing new insights into the biology and therapy of AML.