These findings suggest that for a vertical combination to be synthetic lethal, each inhibitor must target a distinct node of the RAF-MEK-ERK pathway (Figure S2E)
These findings suggest that for a vertical combination to be synthetic lethal, each inhibitor must target a distinct node of the RAF-MEK-ERK pathway (Figure S2E). We performed CDDO-Im Bliss analyses to determine whether the inhibitory activities of each combination were additive or CDDO-Im synergistic; Bliss scores greater than 1.0 indicate synergy. and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of unfavorable feedback and system failures including loss of ERK signaling, functions as an oncogene. Given the >95% mutation frequency in PDAC and substantial experimental evidence that KRAS is essential for PDAC maintenance (Collins et al., 2012; Ying et al., 2012), KRAS is the most attractive target for therapeutic intervention in this disease (Waters and Der, 2018). Despite significant recent progress in developing direct inhibitors of mutant KRAS (Janes et al., 2018; Ostrem and Shokat, 2016), with two now under clinical evaluation, these are selective for KRASG12C, a mutant that is found infrequently (only ~2%) in PDAC (Cox et al., 2014). Inhibitors of KRAS effector signaling remain promising KRAS-targeted therapies (Papke and Der, 2017; Ryan and Corcoran, 2018). Of the multitude of effectors, substantial experimental studies and PDAC patient data support the key role of the RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade in driving KRAS-dependent PDAC growth. Mutationally activated BRAFV600E can phenocopy mutant KRAS and drive the development of invasive and metastatic PDAC (Collisson et al., 2012), and mutations are found in ~50% of the rare PDAC that are wild type (WT) (TCGA, 2017). Further, an effector small interfering RNA (siRNA) screen exhibited that KRAS-dependent cancers are driven largely by RAF (Yuan et al., 2018). These observations support the RAF-MEK-ERK cascade as the key effector pathway driving KRAS-dependent PDAC. However, to date, therapeutic targeting of MEK in KRAS mutant lung cancer demonstrated limited to no efficacy in patients (Blumenschein et al., 2015; J?nne et al., 2017). Challenges to the effective use of inhibitors of ERK MAPK signaling include toxicity in normal cells (Blasco et al., 2011) and adaptive responses to inhibitor treatment, resulting in ERK reactivation and bypass of inhibitor action (Duncan et al., 2012). Another challenge in targeting the ERK MAPK cascade is usually determining which level of the three-tiered kinase cascade will provide the most effective and long-term therapeutic response. At the top of the pathway are the three highly related RAF isoformsARAF, BRAF, and CRAF/RAF1that exhibit distinct functions in RAS-driven cancers (Desideri et al., 2015). BRAF-selective inhibitors caused paradoxical activation of ERK signaling in RAS mutant CDDO-Im cancers (Hatzivassiliou et al., 2010; Poulikakos et al., 2010). Pan-RAF inhibitors (RAFis) overcome paradoxical activation and showed greater activity in mutant cancers (Peng et al., 2015; Yen et al., 2018). However, genetic deletion studies in Kras-driven mouse models argue that pan-RAF inhibition may be limited by normal cell toxicity and that a CRAF-selective strategy may provide a tumor-selective therapy (Blasco et al., 2011; Karreth et al., 2011). In contrast, deficiency in a or (Freeman et al., 2013), we found that suppression of any RAF isoform alone was sufficient to partially impair growth of all six KRAS mutant PDX PDAC cell lines (Figures 1A, S1C, and S1D), demonstrating that each gene contributes to KRAS-dependent PDAC growth, with the general hierarchy of significance CRAF>BRAF>ARAF. This Rabbit polyclonal to Nucleostemin obtaining is similar to that made by McCormick and colleagues, where concurrent siRNA suppression of all three genes was required to cause an comparative suppression of growth of KRAS mutant cell lines as seen with suppression (Yuan et al., 2018). Open in a separate window Physique 1. Concurrent Inhibition of All RAF Isoforms Diminishes PDAC Growth(A) PDAC cell lines were infected by lentivirus vectors encoding nonspecific (NS) control or distinct shRNAs targeting or sequences. Colonies were stained by crystal violet ~10 days after plating. Data are presented as median. All p values shown are in comparison to the vehicle control for the individual graph. Adjusted p values are from Dunnetts multiple comparison test. Adjusted p values: Pa01C (= 0.6792, =.