Abstract 5404: PAK4 allosteric modulators (PAMs) repress the Wnt/β-catenin signaling pathway and tumor growth

Abstract

Abstract Wnt/β-catenin signaling is a ubiquitous pathway conserved throughout evolution and plays a role in embryonic and cancer development. This pathway has proven to be difficult to target therapeutically. Another intractable target is oncogenic Ras which is highly mutated in many cancer types, including pancreatic, colon and lung. An available target at the intersection of both of these genes is p21-Activated Kinase 4 (PAK4). PAK4 (1 of 6 PAK proteins) that is downstream of the Ras oncogene and a direct kinase that stabilizes transcriptional activity of β-catenin. Therefore therapeutically targeting PAK4 in cancer could be beneficial in a broad range of cancer types. PAK4 Allosteric Modulator (PAM) represents a novel and selective class of compounds that inhibit PAK4 allosterically. Methods: Flow cytometry and CellTiter AQueous One assay (MTS) were used to determine compound effects on cell cycle distribution and proliferation. CCLE, COSMIC and other databases were used for bioinformatics analysis of mutations in cancer genes. Deep sequencing and qPCR were used to analyze mRNA expression profiles. Proteomic platforms - KinomeScan, PTScan (Cell Signaling) and immunoblots were used to study phosphorylation signature of whole cell proteins and total protein steady state levels. Results: We identified a selective, orally bioavailable small molecule PAM, KPT-9274, which demonstrated anti-tumor activity in a variety of cancer cell lines (IC50 values from 0.005 - 1 μM). Bioinformatics revealed that sensitivity to KPT-9274 was directly correlated with mutations in APC or N-ras and inversely correlated with mutations in β-catenin, K-ras, or PI3K. KPT-9274 and other PAMs reduced phosphorylation and steady state levels of PAK4 protein while reducing Phospho-S675 and total β-catenin, Wnt5, Phospho- and total LRP6, Dvl2, Phospho-Akt, and Phospho-ERK. PAMs also reduced β-catenin transcriptional activity (i.e. cyclin D1, MM7, and S100A4). PAMs arrested cancer cell cycle at the G1 and G2 phases and induced apoptosis through Caspase and PARP cleavage. KPT-9274 (∼100 mg/kg BIDx5 orally) has demonstrated potent anti-tumor activity against hematological (Z-138, Molt-4, MM1S) and solid (MDA-MB-468 and Colo-205) xenograft models in mice. PAM-treated xenografts showed reduction of PAK4, β-catenin and cyclin D1 proteins. Conclusions: PAK4 represents a novel anti-cancer target at the crossroads of Ras and Wnt/β-catenin signaling. We have identified selective small molecule PAMs with anti-tumor activity both in vitro and in vivo. These allosteric modulators induce tumor cell growth arrest and apoptosis. Bioinformatics helped identify potential predictive markers in the Wnt signaling pathway while deep sequencing and proteiomics revealed possible PDn markers. Based on the in vitro and in vivo activity, KPT-9274 may be beneficial for the treatment of a wide variety of cancers and preclinical toxicology studies are ongoing. Citation Format: William T. Senapedis, Scott Donovan, Gali Golan, Dilara McCauley, Joel Ellis, Marsha Crochiere, Trinayan Kashyap, Boris Klebanov, Sharon Shacham, Yosef Landesman, Erkan Baloglu. PAK4 allosteric modulators (PAMs) repress the Wnt/β-catenin signaling pathway and tumor growth. [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 5404. doi:10.1158/1538-7445.AM2015-5404