Abstract
Leukemia involving rearrangements of the MLL gene is resistant to standard therapies and is often a fatal disease. MLL gene-rearrangements are commonly associated with infant-leukemia and secondary leukemias. New therapeutic strategies are needed to achieve longer survival and improve cure rates in patients with these and other refractory leukemias. Toll-like receptor (TLR) agonists are known as potent immune stimulatory agents that can elicit host anti-tumor effects in murine tumor models. We hypothesized that targeting TLRs expressed on leukemia cells with TLR agonists may have direct antitumor effects against leukemia cells. In this study, we investigated the effects of TLR agonists specific for TLR3, 4, 7, and 9, (i.e., polyinosine-polycytidylic acid (Poly(I:C)), monophosphoryl lipid A (MPL), imiquimod (IMQ), and CpG oligodeoxynucleotides (CpG ODN)), in MLL-AF9 knock-in mice that develop myeloid leukemia akin to human MLL-AF9 disease. In contrast to Poly(I:C), MPL, and CpG ODN, treatment of MLLAF9 cells with TLR7 agonist IMQ significantly increased the surface expression of CD40, CD54, CD80, and CD86 on MLL-AF9 cells in vitro. TLR7 mRNA and protein expression in MLL-AF9 cells were confirmed by real-time RT-PCR and intracellular staining/FACS analysis. Most importantly, TLR7 agonist strongly inhibited the in vitro MLL-AF9 cells in a drug dose- and treatment time-dependent manner. Whereas MLL-AF9 cells proliferated rapidly in culture with more than 40-fold increase of cell number in 5 days, the addition of IMQ at 5 mcg/ml fully inhibited the growth and induced profound apoptosis of MLL-AF9 cells with less than 2% of leukemia cells left at day 5 of culture. IMQ-mediated apoptotic death of MLL-AF9 was confirmed by viable cell counts, TMRE staining, Western blots and intracellular staining of the cleavage of caspases and PARP. Preincubation of MLLAF9 cells with caspases 8 and 10 inhibitors effectively blocked IMQ-induced apoptosis and sustained the proliferation of leukemia cells in cultures. To further determine the intracellular pathways engaged by IMQ, microarray gene expression profiles of 24-hour IMQ-treated vs. untreated MLL-AF9 cells were compared. Gene Set Enrichment Analysis (GSEA) showed that IMQ treatment resulted in up-regulated expression of a set of proapoptotic genes (e.g., p53, Bax, caspase 8, Apaf-1, etc) involved in apoptotic pathways. To determine whether IMQ pre-treatment of MLL-AF9 cells would prolong survival due to an apoptotic effect, cohorts of NOD-scid IL2Rgamma mice were i.p. injected with a lethal dose of MLL-AF9 cells with or without pre-incubation with IMQ. Mice receiving 5×106 untreated MLL-AF9 cells resulted in uniform lethality in 4 weeks. In contrast, mice receiving the same lethal dose of MLL-AF9 cells pretreated with IMQ had a significant prolonged survival, confirming in vitro findings that IMQ-treated MLL-AF9 cells undergo apoptosis. Administration of IMQ (daily i.p. injection at 1 mg/kg for 5 days) strongly inhibited leukemia cells growth and significantly prolonged the survival time of MLLAF9 mice. Flow cytometry results confirmed that residual MLL-AF9 cells recovered from IMQ treated mice were apoptotic and had up-regulated expression of cleaved caspases and PARP. In summary, our results demonstrate that TLR7 targeting of MLL-AF9 cells can directly induce apoptosis of MLL-AF9 cells in vitro and in vivo, providing new insights into the TLR-targeted therapy of refractory or relapsed leukemias.
Macrophage activation by edible mushrooms is due to the collaborative interaction of toll-like receptor agonists and dectin-1b activating beta glucans derived from colonizing microorganisms
