Abstract 5492: Functional characterization of human toll-like receptor 5 (TLR5) genetic variants

Abstract

Abstract Toll-like Receptor 5 (TLR5) is a type 1 transmembrane receptor involved in the first line of defense against invading bacterial pathogens expressing flagellin. TLR5 is expressed on a wide variety of tumors and induction of TLR5 signaling exhibits anti-tumor activities via mobilization of innate and subsequent adaptive antitumor immune response. Entolimod, a pharmacologically optimized flagellin of Salmonella, is the first TLR5 agonist to enter clinical trials. This agent binds to TLR5 and activates NF-kB and other signaling pathways to elicit antitumor activity. The antitumor activity of entolimod has been shown to be dependent on the expression of functional TLR5 on tumors. Since TLR5 is genetically polymorphic, we hypothesized that variations in the gene would alter responses to therapy with entolimod. We therefore characterized genetic variants of TLR5. Expression constructs for nine naturally occurring human TLR5 variants namely Thr82Ile (Ile82): c.245C>T [rs764535]; Asn143Thr (Thr143): c.428A>C [rs5744167]; Arg149Cys (Cys149): c.445C>T [rs201906412]; Gln387Ter (Ter387 or 387*): c.1159C>T [rs147164861]; Arg392Ter (Ter392 or 392*): c.1174C>T [rs5744168]; Leu487Ile (487Ile): c.1459C>A [rs5744171]; Asn592Ser (Ser592): c.1775A>G [rs2072493]; Phe616Leu (Leu616): c.1846T>C [rs5744174] and Glu731Asp (Asp731): 2193A>T [rs148986834], were created from C-terminal FLAG-tagged wild type (WT) TLR5. All constructs together with a construct with no TLR5 insert (empty vector) were transfected in cos-1 cells. In vitro translation of the constructs in rabbit reticulocyte lysates (RRL) were also performed. Cos-1 cells transfected with constructs were also treated with flagellin and entolimod and with proteasome and autophagy inhibitors. Recombinant and synthesized proteins were examined by western blot analysis and NF-kB activity was measured using a Dual-Glo Luciferase Assay. The molecular weight of the recombinant proteins was approximately 100kD. There were variations in expression levels of immunoreactive proteins (10-80% of WT) and the NF-kB activity induced was lower or similar to WT. The Ile82 variant showed a significant reduction in levels of immunoreactive protein (10% of WT) which correlated with decreased NF-kB activity compared to WT, after stimulation. All proteins synthesized in RRL had similar molecular weight except Ter387 and Ter392 which expressed truncated proteins of approximately 40kD and had no NF-kB activity compared to the WT. The Ile82 protein was degraded primarily through the autophagy pathway. The mRNA and cytokine levels of the variant constructs will be reported. These results suggest that genetic variations in the TLR5 gene may be predictive of response to entolimod. This predictive biomarker will be explored in the ongoing phase I study of entolimod. Citation Format: Andrzej J. Wierzbicki, Araba A. Adjei, Nuttapong Ngamphaiboon, Thanyanan Reungwetwattana, Andrei V. Gudkov, Alex A. Adjei. Functional characterization of human toll-like receptor 5 (TLR5) genetic variants. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5492. doi:10.1158/1538-7445.AM2015-5492