J. A. Castellanos1, M. Van Saun2, N. Nagathihalli2, Y. Xiong1, C. Kasmai3, N. Merchant2 1Vanderbilt University Medical Center,Surgical Sciences,Nashville, TN, USA 2University Of Miami,Surgery,Miami, FL, USA 3Meharry Medical College,Nashville, TN, USA
Introduction:
KRAS is mutated in ~95% of pancreatic ductal adenocarcinoma (PDAC) and is the initiating genetic event in the development of this lethal cancer. Despite great efforts, we have been unable to effectively target KRAS and it is felt that Ras proteins are ‘undruggable’. We now propose a novel approach to overcome this resistance by targeting MEK and CDK 4/6, two key downstream effectors of KRAS.
Methods:
Expression of Retinoblastoma (Rb) and ERK protein levels in KRAS wild-type and mutant human PDAC cell lines was determined at baseline and with MEK (MEK162) and/or CDK4 (LEE011) inhibition. The effects of combined therapy on cell-cycle progression, colony formation, and invasion were determined. Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) mice were treated with CDK4/6 and MEK inhibition and transcriptomic profiles were obtained by performing RNAseq on PKT mouse tumors after 2 weeks of treatment with vehicle, LEE011, MEK162, or the combination in addition to wild type Ptf1acre/+;LSL-KrasWT/WT;Tgfbr2flox/flox. Differentially expressed genes (DEGs) were identified using baySeq, DESeq2, and EdgeR in a pairwise fashion, and the lists of genes were inputted into WebGestalt (http://bioinfo.vanderbilt.edu/webgestalt/) for pathway enrichment analysis using gene ontology (GO), KEGG, and Wikipathways. Gene set enrichment analysis (GSEA) was performed to predict therapeutic targets and assess therapeutic response.
Results:
The CDK4/6 inhibitor LEE011 effectively inhibits phosphorylation of Rb in PDAC cell lines. Combined inhibition of CDK4/6 and MEK decreases pRB and pMAPK expression, delays cell cycle progression, decreases colony formation, and decreases invasion in the KRAS mutant cell lines. GSEA of wild type mouse pancreata vs. control mouse tumor revealed significant upregulation in the MYC targeted pathway, a key regulator of Ras mediated therapeutic resistance. Overall survival (OS) in PKT mice treated with MEK162 or LEE011 alone was not significantly different compared to controls, but mice receiving combined CDK4/6 and MEK inhibition exhibited a 400% increase in OS (59 vs. 251.5 days). Combined CDK4/6 and MEK inhibition significantly downregulated IL-6/STAT3, Kras, EMT, and IL-2/STAT5 related pathways compared with control mice. Combination treated mice also had significant downregulation in IL-6/STAT3 signaling compared to MEK treated mice.
Conclusions:
Targeting KRAS in PDAC through downstream inhibition of MEK remains ineffective due to upregulation of STAT3 signaling. Targeting two key downstream effectors of KRAS signaling with combined inhibition of CDK4/6 and MEK overcomes STAT3 mediated chemoresistance and results in significantly enhanced therapeutic efficacy in the aggressive PKT genetic mouse model of PDAC.