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. 2011 May 24;108(21):8773-8.
doi: 10.1073/pnas.1105941108. Epub 2011 May 9.

Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress

Alice T Shaw  1 , Monte M Winslow, Margaret Magendantz, Chensi Ouyang, James Dowdle, Aravind Subramanian, Timothy A Lewis, Rebecca L Maglathin, Nicola Tolliday, Tyler Jacks
Affiliations

Selective killing of K-ras mutant cancer cells by small molecule inducers of oxidative stress

Alice T Shaw et al. Proc Natl Acad Sci U S A. .

Abstract

Activating K-RAS mutations are the most frequent oncogenic mutations in human cancer. Numerous downstream signaling pathways have been shown to be deregulated by oncogenic K-ras. However, to date there are still no effective targeted therapies for this genetically defined subset of patients. Here we report the results of a small molecule, synthetic lethal screen using mouse embryonic fibroblasts derived from a mouse model harboring a conditional oncogenic K-ras(G12D) allele. Among the >50,000 compounds screened, we identified a class of drugs with selective activity against oncogenic K-ras-expressing cells. The most potent member of this class, lanperisone, acts by inducing nonapoptotic cell death in a cell cycle- and translation-independent manner. The mechanism of cell killing involves the induction of reactive oxygen species that are inefficiently scavenged in K-ras mutant cells, leading to oxidative stress and cell death. In mice, treatment with lanperisone suppresses the growth of K-ras-driven tumors without overt toxicity. Our findings establish the specific antitumor activity of lanperisone and reveal oxidative stress pathways as potential targets in Ras-mediated malignancies.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of tolperisone and tolperisone-like drugs as synthetic lethal small molecule inhibitors of K-rasG12D-transformed MEFs. (A) Schematic of high-throughput small molecule library screening. (B) Representative data from primary screening. The viability score is the normalized CTG luminescent value associated with each compound. A viability score of 1.0 indicates that there is no difference in viability relative to DMSO-treated cells. (C) Viability of wild-type and K-rasG12D-expressing MEFs across a range of tolperisone concentrations, as assessed using the CTG assay. Mean ± SEM of three independent lines per genotype, each plated in triplicate. (D) Cell number after tolperisone treatment of wild-type and K-rasG12D MEFs, as assessed using a BrdU cytoblot assay. Mean ± SEM of three independent lines per genotype, each plated in triplicate. (E) Chemical structures of tolperisone and the tolperisone-like drug LP. (F and G) Dose–response experiment testing the effects of LP on cell viability in K-rasG12D (F) or K-rasG12D;p53−/− MEFs. Mean ± SEM of three independent lines per genotype, each plated in triplicate. For all dose–response experiments shown, assays were performed 48 h after compound addition.
Fig. 2.
Fig. 2.
Lanperisone induces cell death of oncogenic K-ras–expressing MEFs. (A) Morphological changes induced by LP treatment of K-rasG12D-expressing MEFs. Phase contrast (Upper) and Oregon Green 488 phalloidin fluorescence (Lower) are shown. (B) Enhanced cell killing of K-rasG12D-expressing MEFs compared with wild-type MEFs, as assessed by FACS for DNA content. Percentages are the mean ± SD of three independent MEF lines. (C) Cell cycle analysis of LP vs. DMSO-treated MEFs. Percentages are the mean ± SD of three independent MEF lines. (D) FACS for PI. Percentages are the mean ± SD of three independent MEF lines. For all experiments shown, K-rasG12D-expressing MEFs were derived from K-rasLSL-G12D;Mox2-Cre embryos. Cells were treated with LP or DMSO for 6 h. Similar results were observed with K-rasG12D;p53−/− MEFs.
Fig. 3.
Fig. 3.
Lanperisone-mediated induction of intracellular reactive oxygen species and oxidative cell death in K-ras mutant MEFs. (A) FACS of DCF-DA stained MEFs. MEFs were treated with 10 μM LP for 6 h. (B) High level ROS induction as a function of LP dose, as shown by DCF-DA staining and FACS analysis. MEFs were treated with the indicated concentrations of LP for 6 h. Values shown are mean ± SD of triplicate wells. (C) Time course of LP-mediated ROS induction and cell death, as assessed by FACS and costaining for DCF-DA and PI. Percent of cells in each gate is shown. (D) Suppression of LP-mediated cell death by pretreatment with antioxidants, as shown by FACS. K-rasG12D;p53−/− MEFs were pretreated and then exposed to 20 μM LP for 6 h. (E) Suppression of LP-induced death by cobalt chloride, even at high doses of LP. Cells were treated with LP for 6 h. Data are representative of three independent K-rasG12D MEF lines. (F) MEK-dependent, translation-independent cell killing by LP (10 μM, 6 h). Data are the mean ± SD of triplicate wells and is representative of three independent MEF lines.
Fig. 4.
Fig. 4.
Lanperisone inhibits tumor growth in vivo. Fully transformed, compound mutant K-rasG12D;p53−/− MEFs were injected s.c. into the flanks of nude mice. Tumor-bearing mice were then treated with either vehicle (dH2O) or LP. (A) Body weights before and after treatment in control and LP-treated mice. Mean ± SEM is shown. ns, not significant. (B) Initial estimated volumes of s.c. tumors before treatment. Mean ± SEM is shown (n = 23/cohort). (C) Final tumor weights of s.c. tumors after 7 d of control or LP treatment. Mean ± SEM is shown (n = 23/cohort).
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