APC and P53 mutations synergise to create a therapeutic vulnerability to NOTUM inhibition in advanced colorectal cancer

Abstract

Objective: Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with the majority of cases initiated by inactivation of the APC tumour suppressor. This results in the constitutive activation of canonical WNT pathway transcriptional effector ß-catenin, along with induction of WNT feedback inhibitors, including the extracellular palmitoleoyl-protein carboxylesterase NOTUM which antagonises WNT-FZD receptor-ligand interactions. Here, we sought to evaluate the effects of NOTUM activity on CRC as a function of driver mutation landscape.

Design: Mouse and human colon organoids engineered with combinations of CRC driver mutations were used for Notum genetic gain-of-function and loss-of-function studies. In vitro assays, in vivo endoscope-guided orthotopic organoid implantation assays and transcriptomic profiling were employed to characterise the effects of Notum activity. Small molecule inhibitors of Notum activity were used in preclinical therapeutic proof-of-principle studies targeting oncogenic Notum activity.

Results: NOTUM retains tumour suppressive activity in APC-null adenomas despite constitutive ß-catenin activity. Strikingly, on progression to adenocarcinoma with P53 loss, NOTUM becomes an obligate oncogene. These phenotypes are Wnt-independent, resulting from differential activity of NOTUM on glypican 1 and 4 in early-stage versus late-stage disease, respectively. Ultimately, preclinical mouse models and human organoid cultures demonstrate that pharmacological inhibition of NOTUM is highly effective in arresting primary adenocarcinoma growth and inhibiting metastatic colonisation of distal organs.

Conclusions: Our findings that a single agent targeting the extracellular enzyme NOTUM is effective in treating highly aggressive, metastatic adenocarcinomas in preclinical mouse models and human organoids make NOTUM and its glypican targets therapeutic vulnerabilities in advanced CRC.

Keywords: COLORECTAL CANCER; ONCOGENES.

Figure 1

Notum is a cell-autonomous oncogene in colorectal adenocarcinoma. (A) UpSet plot showing common differentially expressed genes (p<0.05, log2FC>1) in tumour cells from mouse models of CRC relative to normal colonic epithelium. APKS: adenocarcinoma resulting from orthotopic implantation of Apc/Trp53/Kras/Smad4 quadruple-mutant tumouroids into syngeneic mouse colon. Azoxymethane/dextran sodium sulfate (AOM/DSS): inflammation-induced colon adenocarcinoma. Apc^Min/+ : colon adenoma induced by spontaneous loss of Apc heterozygosity in the Apc^min/+ mouse model. Apc LOF: colonic epithelium after acute loss-of-function of Apc in an Apc^flox/flox::Villin-CreER mouse 5 days after tamoxifen-induced Apc inactivation. (B) Uniform manifold approximation projections (UMAP) of single cell RNA sequencing (scRNAseq) datasets from mouse tumour models in (A), with epithelial/carcinoma cells expressing Epcam (top). Notum expression (bottom) is restricted to epithelial/carcinoma cells (circled). (C) Human NOTUM expression in the Cancer Genome Atlas (TCGA) colon (COAD) and rectal (READ) adenocarcinoma datasets. (D) Left: UMAP representation the cell populations of human CRC primary tumour and adjacent normal epithelium. Cells are coloured according to their cell types. (n=17). Right: UMAP showing NOTUM expression restricted to human carcinoma cells. (E) Western blot analysis for NOTUM in mouse APKS tumouroids infected with control sgRNA (sgCtl) or Notum sgRNA(sgNotum). β-ACTIN was used as a loading control. (F) Images of mouse APKS tumouroids from (E) 3 days after seeding (n=3 technical replicates, with one representative of three independent experiments shown). Scale bar: 200 µm. (G) Clonal seeding efficiency of single APKS cells from (E/F). (H) MTT proliferation assays of APKS mouse tumouroids (n=3 technical replicates, with one representative of three independent experiments shown). (I) Left: Primary tumours formed 8 weeks after orthotopic injection of APKS tumouroids into the distal colon of syngeneic recipient mice (n=5 in sgCtl group, n=4 in sgNotum group). Right: Weight of primary tumours from the left. (J) Left: Livers of the mice bearing primary APKS tumours from (I). White asterisks show metastatic liver lesions. Right: The number of macrometastases visible in the liver of mice from (I). For all panels: *p<0.05, **p<0.01, ***p<0.001, Student’s t-test. See online supplemental figures 8,9 for validation of driver mutations and sgRNA knockdown. AOM/DSS, azoxymethane/dextran sodium sulfate; CRC, colorectal cancer.

Figure 2

NOTUM has potent tumour suppressive activity in normal and APC-mutant mouse colon tumouroids. (A) Clonal seeding efficiency of wild-type (WT) mouse colon organoids infected with control sgRNA (sgCtl) or Notum sgRNA (sgNotum) (n=3 technical replicates). (B) Images of WT mouse organoids infected with control sgRNA (sgCtl) or Notum sgRNA (sgNotum) 4 days after seeding (n=3 technical replicates). Scale bar: 100 µm. (C) QRT-PCR analysis of canonical Wnt/B-CATENIN pathway target genes including Axin2, Ascl2, Ccnd1 and Lgr5 in organoids from (B). (D) MTT proliferation assays of WT organoids infected with empty lentiviral vector (EV) or Notum overexpression vector (OE) (n=3 technical replicates). (E) Relative expression level of Wnt pathway target genes including Axin2, Ascl2, Ccnd1 and Lgr5 in WT organoids infected with EV or Notum OE (n=3 technical replicates, with one represent). (F) Images of Apc mutant mouse tumouroids infected with EV or vector overexpressing Notum 4 days after seeding (n=3 technical replicates, with one representative of three independent experiments shown). Scale bar: 200 µm. (G) Seeding efficiency of Apc mutant tumouroids from (F). (H) MTT proliferation assays of Apc mutant tumouroids from (F) (n=3 technical replicates, with one representative of three independent experiments shown). (I) Images of Apc mutant mouse tumouroid cultures infected with control sgRNA (sgCtl) or Notum sgRNA (sgNotum) 4 days after seeding (n=3 biological replicates). Scale bar: 200 µm. (J) The percentage of cells in S-phase (EdU+) in cultures from (I). (K) Clonal seeding efficiency of cells from tumouroids in (I). (L) Percent lumen occlusion was measured after orthotopic implantation of Apc-mutant tumouroids infected with control sgRNA (sgCtl) or Notum sgRNA (sgNotum) (n=4 biological replicates). (M) Weight of primary, orthotopic Apc mutant mouse colon tumours with or without Notum loss of function (n=4 biological replicates). (N) The number of macroscopically observable liver metastases in mice harbouring orthotopic from (L, M). For all panels: *p<0.05, **p<0.01, ***p<0.001, Student’s t-test. See (online supplemental figure 8,9) for validation of driver mutations, sgRNA knockdown and overexpression.

Figure 3

Tumour suppressive and oncogenic functions of NOTUM require catalytic activity and occur via paracrine/juxtacrine signalling. (A) Brightfield images of Apc-mutant mouse tumouroids infected with empty vector (EV), vector expressing wild-type NOTUM (OE) or catalytically dead NOTUM with Ser239Ala mutation (S239A) (n=3 technical replicates). Scale bar: 100 µm. (B) Clonal seed efficiency from single cells in (A). (C) S-phase/EdU incorporation assays in tumouroids from (A). (D) Brightfield images of the mouse APKS subclone with hypomorphic NOTUM mutation from online supplemental figure 1N,O (APKS^Hypo ) infected with EV, vector expressing wild-type NOTUM (OE), or catalytically dead NOTUM with Ser239Ala mutation (S239A) (n=3 technical replicates). Scale bar: 100 µm. (E) Clonal seed efficiency from single cells in (D). (F) EdU incorporation assays in APKS^Hypo organoid from (D). (G) Images of APKS^Hypo mouse tumouroids, with tumouroids infected with an mCherry-expressing vector and cocultured with tumouroids infected with a vector expressing GFP only (EV) or expressing GFP and NOTUM (OE) (n=3 technical replicates). Scale bar: 100 µm. (H) EdU assays in the cultures in (G), quantifying the percentage of cells in S phase in the GFP+or mCherry+populations or the overall population. *p<0.05,**p<0.01, ***p<0.001, Student’s t-test. see online supplemental figures 8,9 for validation of driver mutations, sgRNA knockdown and overexpression.

Figure 4

APC and P53 loss synergise to convert NOTUM from a tumour suppressor to an oncoprotein. (A) Relative Notum transcript levels in WT mouse colon organoids, and mouse tumouroids harbouring Apc loss of function (A), Trp53 loss of function (P), both (AP), AP with Kras^G12D mutation (APK) and APK with Smad4 loss of function (APKS) (n=3 technical replicates). (B) Brightfield image of A, P and AP mouse tumouroids infected with control sgRNA (sgCtl) or Notum sgRNA (sgNotum) (n=3). Scale bar: 100 µm. (C) Clonal seeding efficiency of A, P and AP cells from (B). (D) S-phase progression (EdU+) assays in A, P and AP tumouroids from (b) (n=3 technical replicates with one representative of three independent experiments shown). (E) Clonal seeding efficiency of A, P and AP cells infected with NOTUM overexpressing lentivirus (OE) or empty vector control (EV) (n=3 technical replicates). (F) S-phase progression (EdU+) assays in A, P and AP tumouroids as in (E). (G) Analysis of disease-free survival (DFS) in human colon and rectal adenocarcinoma patients (COAD, READ) for which gene expression data is available in the Cancer Genome Atlas (TCGA). Left panel shows DFS for APC mutant, TP53 wild-type patients (n=46), binned on highest and lowest quartile of NOTUM expression (Q1 vs Q4). Right panel shows DFS for APC/TP53 double mutant COAD/READ patients (n=93), binned on highest and lowest quartile of NOTUM expression (Q1 vs Q4). (H) Principal component analysis (PCA) of transcriptome profiles from Apc mutant or Apc/Trp53 mutant mouse cultures with or without shRNA-mediated knockdown of Notum (n=4 biological replicates). (I). Gene set enrichment analysis of transcriptome profiles from (H). For all panels: *p<0.05, **p<0.01, ***p<0.001, Student’s t-test. See online supplemental figure 8,9 for validation of driver mutations, shRNA/sgRNA knockdown and overexpression.

Figure 5

Glypicans mediate the differential effects of NOTUM activity in Apc mutant vs Apc/Trp53 mutant mouse tumouroids. (A) Brightfield image of Apc mutant mouse tumouroids (A) and Apc/Trp53 double mutant tumouroids (AP) infected with control sgRNA (sgCtl), Gpc1 sgRNA (sgGpc1), Gpc4 sgRNA (sgGpc4), Gpc6 sgRNA (sgGpc6) (n=3 technical replicates). Scale bar: 100 µm. (B) EdU assays quantifying fraction of cells in S-phase from cultures shown in (A) (n=3 technical replicates). (C) Western blotting in Apc mutants infected with control shRNA (shCtl) or Notum shRNA (shNotum). β-ACTIN was used as a loading control. Note multiple β-ACTIN blots, one for each of three identical replicate blots. (D) Western blotting in Apc/Trp53 mutant tumouroids infected with control shRNA (shCtl) or Notum shRNA (shNotum). β-ACTIN was used as a loading control. (E) Western blotting for GPC1 (left) and GPC4 (right) using N-terminal antibodies recognising the extracellular domain of indicated glypicans in the media supernatant (Med) or cell lysate (Lys) of Apc mutant or Apc/Trp53 double mutant tumouroids infected with control shRNA (shCtl) or Notum shRNA (shNotum). β-ACTIN was used as a loading control. (F) Western blotting in Apc mutant tumouroids infected with control sgRNA (sgCtl) or Gpc1 sgRNA (sgGpc1). β-ACTIN was used as a loading control. (G) Western blotting in Apc/Trp53 mutant tumouroids infected with control sgRNA (sgCtl) or Gpc4 sgRNA (sgGpc4). β-ACTIN was used as a loading control. (H) Coimmunoprecipitation of IGF1Rb with anti-GPC1 antibody in Apc mutant tumouroids in the presence or absence of the small molecule NOTUM inhibitor ABC99. (I) Coimmunoprecipitation of TGFβR1 with anti-GPC4 antibody in Apc/Trp53 double mutant tumouroids in the presence or absence of the small molecule NOTUM inhibitor ABC99. (J) Western blotting assessing non-canonical TGFβ-TAK1-p38a pathway activity in APKS tumouroids in response to NOTUM inhibition. β-ACTIN was used as a loading control. (K) AP and APKS tumouroids treated with the p38 inhibitor SB202190, with EdU-based quantification of S-phase (n=3 technical replicates). Scale bar: 100 µm. For all panels: **p<0.01, ***p<0.001, Student’s t-test. See online supplemental figures 8,9 for validation of driver mutations, shRNA/sgRNA knockdown and overexpression.

Figure 6

Small molecule inhibition of NOTUM inhibits tumour growth and metastasis in a mouse model of colorectal cancer. (A) Brightfield image of Apc mutant and APKS mouse tumouroids treated with the small molecule inhibitor of NOTUM, ABC99 or vehicle control (DMSO) (n=3 technical). Scale bar: 100 µm. (B) Single cell clonal seeding efficiency and EdU assay quantifying fraction of cells in S phase from tumouroids in (A) (n=3 technical replicates). (C) Bioluminescent imaging (BLI) of mice after endoscope-guided orthotopic implantation of APKS mouse tumouroids into the colonic mucosa. Mice were treated with ABC99 or vehicle (corn oil) control after 4 weeks of engraftment and tumour growth. Mice are treated for indicated number of weeks (week 0=4 weeks post-implantation) (n=7 mice). (D) Quantification of luminescence radiance from (C) at indicated time points (n=7 mice). (E–G) Quantification of primary tumour weight (E), number of macrometastases in the liver (F), and number of lymph node metastases (G) in mice from (C) at the termination of the experiment (8 weeks total, 4 weeks post-treatment). (H) Immunofluorescence staining for KI67 and E-CADHERIN in primary tumour and liver metastases from tumours in (C) (n=7 mice). Nuclei are visualised by DAPI staining. Scale bar of top panels: 200 µm. Lower panels: 50 µm. (I) Kaplan-Meier survival analysis for mice harbouring orthotopic APKS tumours as in (C), treated with ABC99 or vehicle (corn oil) control for 16 weeks following an initial 4-week period of engraftment and tumour growth (total experimental time course of 5 months) (n=10 mice). For all panels, **p<0.01, ***p<0.001, Student’s t-test.

Figure 7

Model for NOTUM tumour suppressive-to-oncogenic switch during the adenoma-adenocarcinoma transition. In early adenomas resulting from APC inactivation, NOTUM exerts tumour suppressive activity through cleavage of oncogenic glypican 1 from the cell surface. Here, GPC1 acts as an oncogene by potentiating IGF1R activity upstream of mTORC1, and NOTUM antagonises this oncogenic axis (TOP). In contrast, on transition to adenocarcinoma with inactivation of P53 (and on accrual of additional driver mutations), NOTUM functions as an obligate oncogene by inhibiting tumour suppressive TGFßR activity via inactivating cleavage of Glypican 4 from the cell surface, which normally potentiates TGFßR activity upstream of TAK1/Smad signalling.