Acquired drug resistance prevents cancer therapies from achieving stable and complete responses.1 Emerging evidence implicates a key role for nonmutational drug resistance mechanisms underlying the survival of residual cancer “persister” cells.2-4 The persister cell pool constitutes a reservoir from which drug-resistant tumours may emerge. Targeting persister cells therefore presents a therapeutic opportunity to impede tumour relapse.5 In an earlier report, we found that cancer cells in a high mesenchymal therapy-resistant cell state are dependent on the lipid hydroperoxidase GPX4 for survival.6 Here, we describe the discovery that a similar therapy-resistant cell state underlies the behavior of persister cells derived from a wide range of cancers and drug treatments. Consequently, we show that persister cells acquire a dependency on GPX4. We demonstrate that loss of GPX4 function results in selective persister cell ferroptotic death in vitro and prevents tumour relapse in vivo. These findings support targeting GPX4 as a therapeutic strategy to prevent acquired drug resistance.

Rat sarcoma (Ras) GTPases regulate cell proliferation and survival through effector pathways including Raf-MAPK, and are the most frequently mutated genes in human cancer. Although it is well established that Ras activity requires binding to both GTP and the membrane, details of how Ras operates on the cell membrane to activate its effectors remain elusive. Efforts to target mutant Ras in human cancers to therapeutic benefit have also been largely unsuccessful. Here we show that Ras-GTP forms dimers to activate MAPK. We used quantitative photoactivated localization microscopy (PALM) to analyze the nanoscale spatial organization of PAmCherry1-tagged KRas 4B (hereafter referred to KRas) on the cell membrane under various signaling conditions. We found that at endogenous expression levels KRas forms dimers, and KRas G12D , a mutant that constitutively binds GTP, activates MAPK. Overexpression of KRas leads to formation of higher order Ras nanoclusters. Conversely, at lower expression levels, KRas G12D is monomeric and activates MAPK only when artificially dimerized. Moreover, dimerization and signaling of KRas are both dependent on an intact CAAX (C, cysteine; A, aliphatic; X, any amino acid) motif that is also known to mediate membrane localization. These results reveal a new, dimerization-dependent signaling mechanism of Ras, and suggest Ras dimers as a potential therapeutic target in mutant Ras-driven tumors.Ras dimer | MAPK signaling | cancer | single molecule imaging | superresolution microscopy T he canonical rat sarcoma (Ras) GTPase family members H-, N-, and K-ras are frequently activated in human cancers (1-4) by recurrent point mutations at codons 12, 13, or 61. These mutations result in constitutive binding of Ras to GTP due to impaired GTP hydrolysis (5). Despite nearly identical G-domains, mammalian Ras isoforms serve nonredundant biological roles and exhibit different mutational spectra in human cancers (1,4,6). These functional differences are in part attributed to distinctions in the membranetethering motif at the C-terminal of Ras known as the hyper-variable region [HVR, which includes the "CAAX" (C, cysteine; A, aliphatic; X, any amino acid) motif] (6, 7). Although mechanisms regulating Ras-GTP levels in cells have been examined extensively, details of how Ras organizes and operates on the cell membrane have been elusive. Efforts on targeting mutant Ras to therapeutic benefits in human cancers by inhibiting membrane localization or GTP binding have not been successful, leaving mutant Ras an intractable drug target (8). Hence, identification of new mechanisms that regulate Ras oncogenesis is crucial to combating mutant Ras-driven cancers.Recent studies using immuno electron microscopy (immuno-EM) have implicated a previously unappreciated spatial mechanism in regulating the biological functions of Ras. In particular, Ras proteins were found to form 5-to 8-membered nanoclusters that serve as signaling scaffolds for recruiting and activating downstream effectors such as Raf and PI3K on the cell membr...

K-Ras and H-Ras share identical effectors and have similar properties; however, the high degree of tumor-type specificity associated with K-Ras and H-Ras mutations suggests that they have unique roles in oncogenesis. Here, we report that oncogenic K-Ras, but not H-Ras, suppresses non-canonical Wnt/Ca(2+) signaling, an effect that contributes strongly to its tumorigenic properties. K-Ras does this by binding to calmodulin and so reducing CaMKii activity and expression of Fzd8. Restoring Fzd8 in K-Ras mutant pancreatic cells suppresses malignancy, whereas depletion of Fzd8 in H-Ras(V12)-transformed cells enhances their tumor initiating capacity. Interrupting K-Ras-calmodulin binding using genetic means or by treatment with an orally active protein kinase C (PKC)-activator, prostratin, represses tumorigenesis in K-Ras mutant pancreatic cancer cells. These findings provide an alternative way to selectively target this "undruggable" protein.