Summary/Abstract: Hepatocellular carcinoma (HCC) is a major cause of cancer-related death worldwide including the United States due to late detection and limited effective therapeutic options. It is imperative to obtain a better understanding of the molecular events underlying the progression of HCC, so that rational therapeutic strategies can be designed. The current proposal focuses on a key signaling molecule, Mixed-Lineage Kinase 3 (MLK3), a member of the MAPK Kinase kinase (MAP3K) family to determine its role in HCC. Using a carcinogen-induced HCC model, we found that MLK3 knockout (KO) mice are protected from developing liver cancer and showed reduced proliferation compared to wild-type (WT) mice. MLK3 expression was induced in a panel of human HCC samples. In addition, in cell-based assays, treatment of various HCC cells with the pan-MLK inhibitor CEP-1347 reduced cell survival, promoted cell cycle arrest and apoptosis, suggesting a potential role of MLK3 in mediating these. Furthermore, overexpression of MLK3 increased the expression and release of cytokines, which in turn promoted MLK3 activity, indicating the presence of a potential positive feedback loop between MLK3 and cytokine signaling axis during HCC progression. Based on these preliminary data, we hypothesize that MLK3 represents a novel target involved in mediating liver tumorigenesis via a crosstalk with cytokine signaling, and targeting of MLK3- cytokine axis by the available pharmacological inhibitors might be a viable means of antagonizing HCC progression. The current proposal is aimed towards establishing MLK3’s role in HCC progression, while elucidating the underlying mechanisms by which this is accomplished, to achieve a mechanistic insight towards MLK3-induced HCC. As a proof-of principle, we will examine the feasibility of pharmacological inhibition of MLK3 on HCC progression using preclinical mouse models. The following specific aims are proposed to achieve our goals: (1) Elucidate the molecular mechanism underlying MLK3-mediated HCC progression, 2) Establish that a crosstalk between MLK3 and cytokine axis promotes HCC progression, and 3) Determine whether pharmacological inhibition of MLK3 and cytokine signaling mitigates liver cancer progression. While studies in Aims 1 and 2 are expected to provide a mechanistic insight towards the signaling pathway by which MLK3 promotes HCC and identify potential downstream targets, those in Aim 3 will provide important proof-of-principle preclinical information on the in vivo efficacy of the pharmacological inhibitor of MLK axis (CEP-1347) and IL-11 antagonist in antagonizing HCC progression. Successful outcome of the proposed studies will not only provide newer insights towards understanding the mechanism by which MLK3 mediates HCC progression, but also provide information on the preclinical efficacy of MLK3 inhibitor combination, which can be utilized towards developing effective MLK3-based therapeutic strategies for targeting ...