Targeting IRAK4 Disrupts Inflammatory Pathways and Delays Tumor Development in Chronic Lymphocytic Leukemia
Authors: Giménez Carabaza Neus , Laia Rosich , Ralph Schulz , Morihiro Higashi , Aymerich Marta , Villamor Neus , Delgado Julio , Juan Manel , Lopez-Guerra Monica , Campo Elias , Seiffert Martina , Colomer Dolors 
Published: October 2019

Abstract
Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and the innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediatedby the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 5% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Gene expression profiling of 423 CLL cases, assessed by the computational GSEA analysis tool, identified enrichment of gene sets related to cytokines and inflammation in the subgroup carrying mutations in MYD88. Accordingly, CLL cells from individuals with MYD88 mutations showed higher p65 NF-κB activity and secreted higher basal levels of CCL2, CCL3 and CCL4 cytokines compared with unmutated cases. To assess the effects of this pathway in CLL, we stimulated these cells with a TLR agonists mix, containing Pam3CSK4, HKLM, FSL1 and ODN2006, which was able to induce cytokines secretion, increase in NF-KB activity and STAT3 signaling as well as cells proliferation and migration. Here, we study the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. ND2158 was able to efficiently decrease all TLR effects seen with the mix like STAT3 signaling and the secretion of CCL2, CCL3, CCL4, TNFα, IL1β, IL10 and IL6 cytokines (assessed by Luminex). NF-κB signaling was also modulated by the drug, as observed by the downregulation of IκBα phosphorylation protein level, p65 translocation to the nucleus and decreased p65 DNA binding activity. ND2158 also impaired CLL proliferation (checked by carboxyfluoresceinsuccinimidyl ester staining) and migration towards CXCL12 in MYD88-mutated and -unmutated CLL cases.ND2158 also induced a selective dose-dependent cytotoxic effect in CLL cells, without differences regarding their MYD88 mutational status. To further validate this results, we checked the effect of ND2158 in splenocytes from leukemic Eμ-TCL1 mice and similar antitumor effects were seen.
In addition to the direct impact on tumor cells, we analyzed whether ND2158 also affects other immune cells that also signal via MYD88/IRAK4. Monocytes, broadly known for their importance in tumor supporting, were also modulated by TLR agonists, and a reduction in activation markers and cytokines secretion was achieved after ND2158 treatment.
In vivo studies in Eμ-TCL1 leukemic mice showed a significant reduction in tumor load in CLL-affected organs (peripheral blood, spleen, lymph node and peritoneal cavity) associated with reduced PD-L1 expression levels in mice treated with 100 mg/kg/day of the IRAK4 inhibitor. A decrease in spleen weight and size was also detected after the treatment. Corroborating the in vitro studies,ND2158 also modulated tumor-supporting monocytes. The effect of ND2158 was also assessed in CD8+ effector T cells, where a low proliferative (Ki-67+ percentage), exhausted phenotype (increase in TIGIT, CD244, CD160 and LAG3 markers) and less functional population (increase in the immune checkpoint protein PD-1) was found.
Based on our in vitro data, it seems that ND2158 targets both MYD88 mutated and unmutated CLL cases to a similar degree. Since CLL progression in the TCL1 mouse model has not been reported to be driven by an activating MYD88 mutation, our in vivo results support the notion that IRAK4 inhibition might also be effective in CLL patients with an unmutated MYD88 status. To overcome the potential negative impact of IRAK4 inhibition on T cells, the combination treatment with drugs that improve T cell function and avoid their rapid exhaustion should be considered. As we observed increased expression of several inhibitory receptors on CD8+ T cells in ND2158-treated mice, including PD-1 and TIGIT, blocking these receptors with antibodies might be a successful strategy to overcome loss of T cell function and improve therapy outcome.
In summary, our data show the importance of TLR signaling in CLL development and suggest IRAK4 as a novel treatment target for CLL that targets both the malignant cells and the tumor-supportive inflammatory milieu.

Disclosures
No relevant conflicts of interest to declare.