Abstract 899: Development of Hedgehog acyltransferase antagonists as a novel way to inhibit Shh signaling in pancreatic cancer

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Pancreatic adenocarcinoma is among the leading causes of cancer-related death in the US, with low response to standard therapy and high recurrence rates. Abnormal overexpression of the secreted morphogen Sonic Hedgehog (Shh) has been implicated as an important contributor to pancreatic adenocarcinoma in adults. Shh produced by pancreatic cancer cells drives tumor growth through both autocrine and paracrine mechanisms. The Shh pathway is thus an attractive therapeutic target. Currently there is an unmet need for drugs effective in inhibiting this pathway. In order to signal, Shh must be modified by attachment of the 16-carbon fatty acid palmitate, a reaction mediated by Hedgehog Acyltransferase (Hhat). Here we show that Hhat drives the growth of human pancreatic cancer cells in vitro and in vivo, and report the development of novel small molecule inhibitors of Hhat that can be used as anti-cancer agents. We used lentiviral shRNA vectors to knockdown Hhat in two human pancreatic adenocarcinoma cell lines. Hhat depletion in AsPC-1 cells resulted in decreased autocrine Shh signaling, and a strong reduction in both anchorage-dependent (80%) and anchorage-independent (70%) cell proliferation. In Panc1 cells, Hhat depletion decreased paracrine Shh signaling, as monitored by a co-culture assay of Panc1 cells and C3H10T1/2 mouse fibroblast reporter cells. Hhat knockdown also led to decreased tumor growth in a Panc1 xenograft mouse model in vivo. These findings provide proof of concept that Hhat plays an essential role in pancreatic cancer. In order to identify small molecule inhibitors of Hhat, we employed high-throughput screening (HTS) using an in vitro assay that monitors palmitoylation of a Shh peptide by Hhat. This assay was optimized for analysis by Scintillation Proximity Assay (SPA) technology. A chemical library of 85,000 compounds was screened, 95 hits were validated and 4 lead compounds were selected; structures were confirmed by NMR analysis. Based on its ability to function as a dose dependent inhibitor of Shh palmitoylation in vitro (IC50 = 0.895µM) and in cells, RU-SKI 43 was chosen for further analysis. RU-SKI 43 was shown to be a specific inhibitor of Shh palmitoylation. It does not affect palmitoylation of H-Ras or Fyn, nor does it alter myristoylation of c-Src. Importantly, in the cell-based models of pancreatic cancer, treatment with RU-SKI 43 resulted in a strong decrease in AsPC-1 cell growth (75%), and in Panc1-driven paracrine Shh signaling (80%). A second generation of Hhat inhibitors has been designed based on the structure of RU-SKI 43. One of these compounds, RU-SKI 101, has an IC50 of 156 nM for Hhat inhibition in vitro and is currently being tested as an anti-cancer agent. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 899. doi:1538-7445.AM2012-899