The TLR Adaptor Protein MyD88 Mediates Cell Sensitivity to HDAC Inhibitors through a Cytokine-Dependent Mechanism

Introduction

Histone deacetylase inhibitors (HDI) are promising new agents for the treatment of haematological malignancy including lymphoma. HDI have potent anti-proliferative effects in cell-based studies; in the clinical setting, however, the picture is quite variable, and the outcome of HDI therapy is complex and difficult to predict.

The toll-like receptor (TLR) adaptor protein MyD88 (Myeloid Differentiation Primary Response Gene 88) is expressed at high levels in haematopoietic tissues and has recently been found to be the target for somatic mutation in lymphoid malignancies. In particular, substitution of the amino acid leucine to proline at position 265 (MyD88 L265P mutation) is estimated to be prevalent in over 90% of cases of Waldenstrom's macroglobulinaemia and around 37% of diffuse large B cell lymphoma (DLBCL) of the ABC subtype.

Methods and Results

Using an unbiased genome-wide loss-of-function screen (Fotheringham et al, 2009), MyD88 was identified as a key regulator and candidate biomarker which mediates the susceptibility of cancer cells to HDI: We show here in mechanism of action studies that expression of MyD88 can influence sensitivity of various cancer cell lines to treatment with an HDI. Short-term siRNA silencing of MyD88 reduced HDI sensitivity of U2OS osteosarcoma cells (as assessed by immunoblotting and FACS/propidium iodide staining), whereas over-expression of MyD88 and MyD88 L265P led to enhanced sensitivity.

Given its role in TLR-signalling, we measured NFκB activity and pro-inflammatory cytokine expression, which were shown to be increased (in particular IL6 levels) when MyD88 was stably or transiently overexpressed in U2OS cells; co-expression of mutated MyD88 L265P augmented this effect. In DLBCL lymphoma cell lines of the ABC subtype, those cells expressing endogenous wild-type MyD88 (RIVA) were found to be less sensitive to HDI treatment compared to those cells carrying a heterozygous MyD88 L265P mutation (HBL-1). MyD88-mutated cells again showed higher levels of IL6 expression on cytokine analysis. Transient knockdown of endogenous MyD88 in ABC DLBCL cells resulted in decreased HDI sensitivity, as did disruption of the TLR/MyD88/NFκB signalling pathway by blocking either the TLR receptor, MyD88 aggregation or IL6 secretion. Immunohistochemical analysis of tumour tissue microarrays from patients with DLBCL carried out in conjunction with this work showed higher expression of MyD88 compared to patients with prostate or colorectal carcinoma. Finally, using a mass spectroscopy approach, we were able to show that MyD88 is acetylated, and that it can be directly deacetylated by the cytoplasmic histone deacetylase HDAC6.

Discussion and translational significance

Collectively our findings show that in cultured cells, high levels of MyD88 expression are associated with increased sensitivity to HDAC inhibition, whereas low level expression coincides with decreased sensitivity. We hypothesize that MyD88, in its role as adaptor protein in TLR signalling, plays an important role in regulating the expression and autocrine action of pro-inflammatory cytokines such as IL6 which contribute to the sensitization of cancer cells to HDI. The central role played by MyD88 in this mechanism highlights the importance of TLR signalling and innate immunity, and more generally the inflammatory properties of the extra-cellular tumour microenvironment in mediating the effects of HDAC inhibitors on malignant cells. Therefore, both expression level of MyD88 and MyD88 mutation status could serve as a potential biomarker for HDI sensitivity in patient selection.