The APC tumour suppressor gene is the most commonly mutated gene in colorectal cancer (CRC). Loss of Apc in intestinal stem cells (ISCs) drives aberrant Wnt signalling and adenoma formation in mice 1 . We previously showed that a reduction in WNT-ligand secretion increases the ability of Apc-mutant ISCs to colonise a crypt (fixation) and accelerate tumourigenesis 2 . Here, we investigate key mechanistic processes whereby Apc-mutant cells gain a clonal advantage over wild-type counterparts to achieve fixation. We find that Apc-mutant cells are enriched for transcripts encoding several secreted Wnt antagonists, with Notum being the most highly expressed. Indeed, conditioned medium from Apc-mutant cells suppresses the growth of wild-type organoids in a Notum-dependent manner. Furthermore, Notum-secreting mutant clones actively inhibit the proliferation of surrounding wild-type crypt cells and drive their differentiation, thereby outcompeting them from the niche. Importantly, genetic or pharmacological inhibition of Notum is sufficient to abrogate the expansion of Apcmutant cells and their ability to form intestinal adenomas. Taken together, we demonstrate Notum as a key mediator during the early stages of mutation fixation, which can be targeted to restore wild-type cell competition and thus, offer novel preventative strategies for high-risk patients. MainThe colonic epithelium displays one of the highest mutation rates of all tissues 3,4 , with lossof-function mutations in the APC tumour suppressor considered a key early event in colorectal cancer (CRC) initiation 5 . For a mutation to be maintained within a crypt, it needs to become "fixed", by mutant cells outcompeting wild-type intestinal stem cells (ISC) from the crypt 6,7 .Previous studies revealed that Apc loss (or Kras activation) confer a clonal advantage to ISCs 7,8, increasing their probability of fixation/winning within the crypt and, in the case of Apc mutation, driving adenoma formation. Even though APC-deficient clones have an increased probability of "winning", they can still be stochastically eliminated from the ISC pool i.e. lose.This suggests uncovering the molecular mechanisms by which APC-deficient cells outcompete wild-type cells could lead to novel chemo-preventative approaches.APC is a negative regulator of Wnt signalling that functions as an integral part of the destruction complex, which directs the phosphorylation and degradation of β-catenin 9 . Since Apc-mutant tumours exhibit constitutive Wnt-pathway activation, we first sought to identify genes differentially upregulated in Apc-mutant cells relative to the normal intestinal epithelium.For this, we performed transcriptomic analysis of tumours that develop in VillinCre ER ;Apc fl/+ (hereafter VilCre ER ;Apc fl/+ ) mice following the sporadic loss of the remaining copy of Apc 10 , akin to human CRC 11 . As expected, Wnt-target genes were highly upregulated in these Apcmutant tumours (Extended Data Fig. 1a). The most highly upregulated gene was Notum (Fig. 1a), which encodes a secreted WNT...

Cancer stem cells (CSCs) are considered to be responsible for treatment relapse and have therefore become a major target in cancer research. Salinomycin is the most established CSC inhibitor. However, its primary mechanistic target is still unclear, impeding the discovery of compounds with similar anti-CSC activity. Here, we show that salinomycin very specifically interferes with the activity of K-ras4B, but not H-ras, by disrupting its nanoscale membrane organization. We found that caveolae negatively regulate the sensitivity to this drug. On the basis of this novel mechanistic insight, we defined a K-ras-associated and stem cell-derived gene expression signature that predicts the drug response of cancer cells to salinomycin. Consistent with therapy resistance of CSC, 8% of tumor samples in the TCGA-database displayed our signature and were associated with a significantly higher mortality. Using our K-ras-specific screening platform, we identified several new candidate CSC drugs. Two of these, ophiobolin A and conglobatin A, possessed a similar or higher potency than salinomycin. Finally, we established that the most potent compound, ophiobolin A, exerts its K-ras4B-specific activity through inactivation of calmodulin. Our data suggest that specific interference with the K-ras4B/calmodulin interaction selectively inhibits CSC.

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