241 Small Molecule Inhibitors Xmd8-92 and Pv1019 Inhibit Pancreatic Tumor Xenograft Growth via a DCLK1 Dependent Mechanism

Abstract

Background: XMD8-92 is a small molecule inhibitor of BMK1/MAPK7 kinase activity, a key member of the MAP kinase cascade, which plays an important role in transmitting oncogenic signals in tumorigenesis. Treatment with XMD8-92 has been reported to inhibit breast and ovarian cancer cell proliferation and tumor xenograft growth. It has also been shown that XMD8-92 binds to DCLK1 (formerly known as DCAMKL-1). Recently, CHK2 (checkpoint kinase) inhibitors have been proposed as chemotherapeutic agents in combination with cytotoxic agents. Treatment with PV1019, a potent and selective CHK2 kinase inhibitor resulted in human ovarian and colorectal cancer cell growth inhibition. The putative intestinal and pancreatic stem cell and Tuft cell marker DCLK1 is upregulated in human pancreatic, esophageal, and colon cancers. Knockdown of DCLK1 with siRNA results in the inhibition of several oncogenes and pluripotency factors, including c-Myc, via microRNA related mechanisms. Aim: To determine whether these small molecule inhibitors regulate pancreatic tumor growth by inhibiting DCLK1. Methods: AsPC-1, human pancreatic cancer cell derived tumor xenografts generated in NOD/SCID mice were injected intraperitoneally with XMD8-92 (50 mg/Kg body weight) and PV1019 (10 mg/Kg). siRNA-targeting DCLK1 encapsulated in poly(lactide-co-glycolide) (PLGA)-based nanoparticles (NP-siDCLK1) and injected directly into tumor xenografts was used as a positive control. Total RNAs isolated from the tumor xenografts and cells from the above experiments were subjected to realtime RT-PCR for mRNA analyses of DCLK1, c-Myc, KRAS, Notch1, EMT, pluripotency factors and CDC25c (downstream target of CHK2 kinase). Protein analyses were carried out by Western blot and immunohistochemistry. Results: XMD8-92, PV1019 and NPsiDCLK1 treatment resulted in AsPC-1 tumor xenograft growth arrest. XMD8-92, PV1019 and NP-siDCLK1 treated tumors demonstrated a statistically significant downregulation (~50%) of DCLK1 and its downstream targets including c-Myc, KRAS, Notch1, ZEB1, ZEB2, Snail, Slug, OCT4, SOX2, LIN28, Nanog, KLF4 and CDC25c. We obtained similar results in vitro following treatment with XMD8-92, PV1019 and NP-siDCLK1. Conclusions: These data taken together demonstrate that small molecule kinase inhibitors like XMD8-92 and PV1019 may regulate pancreatic tumor growth by inhibiting DCLK1 function. Furthermore, these results strengthen the notion that inhibition of DCLK1, a potential central oncogenic regulatory protein, is an effective approach for treating human solid tumor cancers including pancreatic cancer.