A major challenge in cancer therapeutics is the identification of cellular drug targets whose inhibition leads to the selective killing of cancer cells while sparing normal cells. Recent advances in mammalian RNA interference (RNAi) technologies have made it possible to systematically interrogate the human genome for genes whose loss of function constitute synthetic lethality either with the oncogenic state or with particular oncogenic mutations (Ngo et al., 2006
; Schlabach et al., 2008
; Silva et al., 2008
). We have developed barcoded, retroviral/lentiviral-based short hairpin RNA (shRNA) libraries targeting the entire human genome to enable genome-wide loss-of-function analysis through stable gene knockdown (Silva et al., 2005
). Our design also allowed us to develop a multiplex screening platform that enables the highly parallel screening of >10,000 shRNAs in a pool-based format using microarray deconvolution (Schlabach et al., 2008
; Silva et al., 2008
). These technological breakthroughs have made it possible to rapidly interrogate the genome for functional vulnerability of cancer cells and here we apply these to the Ras oncogene.
The Ras family of small GTPases are frequently mutated in human cancers [Reviewed in (Karnoub and Weinberg, 2008
)]. Ras is a membrane-bound signaling molecule that cycles between the inactive, GDP-bound state and the active, GTP-bound state. Growth factor receptor signaling promotes GTP loading and activation of Ras, which in turn activates an array of downstream pathways to promote cell proliferation and survival. Among the major Ras effector pathways is the MAP kinase pathway, the PI3-kinase (PI3K) pathway, RalGDS proteins, phospholipase-Cε and Rac. Each of these has been implicated in mediation of Ras oncogenesis. Ras GAPs (GTPase activating proteins) inactivate Ras by stimulating GTP hydrolysis. Oncogenic mutations in Ras are invariably point mutations that either interfere with Ras GAP binding or directly disrupt Ras GTPase activity, locking Ras in a constitutively active, GTP-bound state. Oncogenic mutations have been found in all three members of the Ras gene family with KRAS
being the most frequently mutated. KRAS
mutations are found at high frequencies in pancreatic, thyroid, colon, lung and liver cancers and in myelodyspastic syndrome and are correlated with poor prognosis (Karnoub and Weinberg, 2008
Despite its prominent status as a cancer drug target, therapeutics aimed at disrupting the Ras pathway have proven challenging thus far. Inhibitors of farnesyl transferase, the enzyme that prenylates Ras for its membrane localization, have met with only limited success (Karnoub and Weinberg, 2008
). Chemical screens in isogenic Ras mutant and wild type cell lines have identified compounds that exhibit preferential toxicity towards Ras mutant cells (Torrance et al., 2001
; Dolma et al., 2003
). However, the translation of these chemical screens into clinical practice has been impeded by the challenge in identifying the protein targets of these chemical entities and subsequent drug development. Inhibitors targeting various Ras effecter pathways could also prove efficacious in treating tumors with Ras mutations, as it was recently shown that a combined application of MEK and PI3K/mTOR inhibitors can reduce tumor burden in a mouse model of Ras-driven lung cancer (Engelman et al., 2008
). However, the prevalence of de novo
and acquired drug resistance to other targeted therapies suggests that combinations of multiple therapeutic agents will be required to effectively inhibit malignant progression.
In principle, tumors can be attacked by either reversing the effects of oncoproteins through inhibition (i.e. exploiting oncogene addiction), or by attacking tumor-specific vulnerabilities caused by the oncogenic state, often by inhibiting proteins that are not oncoproteins themselves (i.e. exploiting non-oncogene addiction) (Solimini et al., 2008;Luo et al., 2009
). The inappropriate rewiring of cellular signaling through oncogene activation should result in vulnerabilities that could be exploited for cancer therapies in theory. Since these vulnerabilities are not obvious and cannot be predicted, the most direct approach to their discovery is through genetic exploration. The systematic identification of genes and pathways necessary for the Ras-driven oncogenic state would provide additional drug targets for therapeutic exploration, shed new light on Ras’ mechanisms of action and potentially provide new biomarkers for patient stratification. To this end, we screened our shRNA library for genes whose inhibition constitutes synthetic lethality with the KRAS
oncogene. We identified a functionally diverse set of genes including a number of mitotic proteins and demonstrate that pharmacological inhibitors targeting these mitotic proteins can selectively impair the viability of Ras mutant cells. These findings point to a previously underappreciated role of Ras in mitotic progression and demonstrate that mitotic stress induced by the Ras oncogene might be exploited for therapeutic purposes.