Targeting TLR Signaling in Chronic Lymphocytic Leukemia with a Selective IRAK-4 Inhibitor

2018 edition

Neus Gimenez

Toll-like receptors (TLRs) signaling is important in cell survival and proliferation, and it is overactivated in several autoimmune diseases and some types of mature B-cell lymphoid neoplasms, such as chronic lymphocytic leukemia (CLL) and the activated B-cell (ABC diffuse large B cell lymphoma (DLBCL) subtype. TLR are essential receptors of the innate immune system and mediate inflammatory signals in B cells, leading to NFkB activation through a MYD88-IRAK4 complex formation, so could be a promising therapeutic strategy for these diseases.  The pathway can be targeted with the IRAK4 inhibitor ND2158, a potent and highly effective kinase inhibitor (Ki of 1.2nM).

We have analyzed TLR pathway in CLL cells and the possibility to target CLL cells with the IRAK4 inhibitor ND2158.

Gene expression profile of 423 CLL cases was analyzed, 25 of them carrying mutations in the TLR/MYD88 pathway, and gene sets related to cytokines and inflammation, such as NF╬║B pathway and STAT signaling were enriched in the mutated cases.

CLL samples with and without MYD88 mutations and mononuclear cells obtained from healthy donors were cultured in vitro in the presence of ND2158 (10 to 100 μM) for 48 hours. ND2158 induced a selective and dose-dependent cytotoxic effect in CLL cells compared to B and T cells from healthy donors.  ND2158 was effective in all CLL samples regardless their mutation status. By western blot we observed that ND2158 was able to downregulate the phosphorylation levels of IkBα and STAT3. To further characterize the effect of ND2158, in vitro stimulation of TLR pathway was performed with its agonists. An increase of cytokine secretion was observed when TLR were stimulated. Basal levels of these cytokines and canonical NFKβ activity (p65) were found to be higher in MYD88 mutated CLL cases than in CLL unmutated ones. It was also observed an increase in the B cell p65 activation and in the capacity of CLL cells adhesion/migration after TLR stimulation, which were were reverted by treating the cells with ND2158. After TLR stimulation, an increase in CLL proliferation was observed at 6 days, which was reduced by treating the cells with ND2158.

In vivo studies using the E╬╝ÔÇÉTCL1 adoptive transfer TCL1 mouse model showed a significant reduction in the tumor load in peripheral blood, lymph node, and peritoneal cavity. Levels of PDL1 in B-cells were also downregulated by ND2158. A decrease in spleen weight and size was detected after the treatment. ND2158 impact in tumor microenvironment was also significant for monocytes. Its effect in T cells and other microenvironment cells needs to be further investigated.

In conclusion, our findings support pharmacological inhibition of IRAK4 as a possible therapeutic strategy in CLL cells regardless MYD88 mutational status, indicating that intrinsic MYD88 signals (that modulate N╬║B pathway) have an important role in CLL cells and tumor microenvironment.