Abstract 4688: Overcoming drug resistance and stemness in oncogenic kras driven pancreatic ductal adenocarcinoma through PAK4 inhibition
Authors: Azmi Asfar S. , Senapedis William , Baloglu Erkan , Landesman Yosef , Kauffman Michael , Shacham Sharon , Wu Jack , Aboukameel Amro , Muqbil Irfana , Mohammad Ramzi M. 
Published: August 2015

Background: In spite of the established role of oncogenic Kras in the development and subsistence of Pancreatic Cancer (PC), no clinically viable agents have been developed to suppress this master regulator. Unlike other druggable proteins, Kras lacks the ideal binding pocket for small molecules. Therefore, there is an unmet need to identify novel druggable sites on Kras or to develop agents that target key effector proteins downstream. The p21-activated kinase 4 (PAK4) is a key effector downstream of Rho family GTPases. PAK4 is over-expressed in most PC cell lines tested but not in normal human pancreatic ductal epithelial cells (HPDE). Gene copy number amplification studies in PC patient cohorts confirmed PAK4 amplification. RNA interference of PAK4 suppresses PC cell proliferation making PAK4 an attractive therapeutic target. In collaboration with Karyopharm Therapeutics, we developed novel PAK4 allosteric modulators (PAMs; KPT-7189, KPT-9274, KPT-9307). Methods: Using multiple molecular biology techniques we tested PAMs activity (in the presence and absence of -ve and +ve controls) on a panel of PC cells lines, PAK4 over-expressing Gemcitabine resistant (GEM-R) PC models and highly resistant flow sorted cancer stem cells (CSC). Pancreatic CSC's are triple positive for CD133+CD44+EpCam+ and undergo epithelial-to-mesenchymal transition (EMT). The toxicity and efficacy of PAMs were evaluated in vitro and in sub-cutaneous mouse models of PC.

Results: The novel, orally bioavailable PAMs show anti-proliferative activity in vitro against different PC cell lines (AsPC-1, Colo-357, MiaPaCa-2, L3.6pl and HPAC IC50s &lt;250nM) while sparing HPDE (IC50s 5 fold higher). Cell growth inhibition was concurrent with apoptosis induction and suppression of colony formation in 5 different PC cell lines (not in HPDE). PAMs reduced RNA and PAK4 protein levels along with the inhibition of proliferative and anti-apoptotic signals downstream of PAK4. Co-immunoprecipitation experiments showed disruption of PAK4 complexes (p65, Bcl-2 and vimentin). Confocal, western blot and RT-PCR analysis of PAM-treated CSC spheroids showed reversal of EMT and suppression of stem markers EpCAM, vimentin and snail with re-expression of epithelial phenotype promoter E-cadherin. Additionally PAMs synergize with Gemcitabine and oxaliplatin (CI&lt;1) in vitro. KPT-9274, possesses desirable PK properties and is well tolerated in mice with the absence of any clinical signs of toxicity up to 200 mg/kg oral daily dose. Pre-clinical animal efficacy (as a single agent and in combination with gemcitabine) in a sub-cutaneous, orthotopic (from primary cells) and LSL-K-Ras G12D/+;LSL-Trp53R172H/+;Pdx-1-Cre transgenic mouse models are ongoing.

Conclusions: These proof of concept studies demonstrating the anti-proliferative effects of novel PAK4 allosteric modulators in pancreatic cancer warrant further clinical investigations.

Citation Format: Asfar S. Azmi, William Senapedis, Erkan Baloglu, Yosef Landesman, Michael Kauffman, Sharon Shacham, Jack Wu, Amro Aboukameel, Irfana Muqbil, Ramzi M. Mohammad. Overcoming drug resistance and stemness in oncogenic kras driven pancreatic ductal adenocarcinoma through PAK4 inhibition. [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 4688. doi:10.1158/1538-7445.AM2015-4688