NOTUM from Apc-mutant cells biases clonal competition to initiate cancer

Abstract

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...

Structural Insights into Notum Covalent Inhibition

“… 29 , 30 More recently, Notum has been identified as a key mediator for adenomatous polyposis coli (Apc)-mutated tumor cell fixation and tumor formation, while Notum inhibitors abrogate the ability of Apc-mutant cells to expand. 31 These pieces of evidence highlight the importance of Notum as a novel target for drug discovery.…”

Lef1 restricts ectopic crypt formation and tumor cell growth in intestinal adenomas

“…Although Apc- mutant adenomas are independent of Wnt ligands ( 15 , 58 ), we found that several Wnt antagonists were decreased after Apc ; Lef1 deletion. This observation may be functionally significant, as a recent study showed that the secreted Wnt antagonist Notum produced by the Apc -mutant adenomas inhibits Wnt signaling in the neighboring WT ISCs ( 59 , 60 ). We found that LEF1 expression was restricted to ligand-independent CRCs, whereas it was not expressed in the ligand-dependent CRCs.…”

Colorectal Cancer Stem Cells: An Overview of Evolving Methods and Concepts

“…In this frame, two studies recently described the capability of Apc-mutated adenoma cells to inhibit stem cell activity of non-mutated cells within the same crypt and adjacent crypts through secretion of soluble Wnt antagonists [ 107 ]. Among these factors, a prominent role is played by NOTUM, whose pharmacological inhibition blocks adenoma formation [ 28 ]. Other important information has been provided by an innovative tracking system, the Confetti-derived Red2Onco, by which oncogenes such as mutated KRAS or PI3K are inserted only in RFP+ tumor cells.…”

“… The timetable of landmark studies that have contributed to defining cCSC biological features and functions. Numbers in parentheses indicate references [ 5 , 6 , 9 , 12 , 17 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. …”