Introduction

Mutations of Adenomatous polyposis coli (APC) are among the most frequent molecular aberrations in colorectal cancer (CRC) and result in oncogenic activation of the Wnt signaling pathway. Previous work showed that despite harboring these mutations, CRC cells require the secretion of Wnt ligands (WNTs) for survival. So far, no systematic analysis to assess the expression and function of all WNTs in CRC has been conducted.

Aims & Methods

We sought to systematically assess the expression of all 19 WNTs in different CRC models. This was performed by bioinformatical analysis of publicly available datasets as well as qRT-PCR and Western blot analysis of CRC organoid lines. Among the highly expressed WNTs, we aimed to investigate their molecular regulation as well as specific oncogenic functions. Employing CRISPR/Cas9 gene editing, we modeled frequent oncogenic mutations in CRC cells and human colon organoids to examine their effect on the expression of WNTs. In order to examine the specific oncogenic effects of selected WNTs, we performed shRNA-based gene knockdown in CRC cell lines and organoids. Using colony formation and CellTiter-Glo (CTG) assays, we assessed the viability of CRC cells and organoids, respectively.

Results

Among all WNTs, especially WNT11 is highly expressed in publicly available datasets and a panel of CRC organoids. Furthermore, the expression of WNT11 is correlated to a signature of high Wnt activation in publicly available datasets of CRC tissues. Introduction of APC mutations through CRISPR/Cas9 in CRC cell lines and human colon organoids resulted in upregulation of WNT11. Inhibition of the downstream transcriptional coactivator of Wnt signaling β-Catenin by small molecule inhibitors and siRNA abrogates the expression of WNT11 in CRC cells and organoids. Knockdown of WNT11 impairs the ​colony formation of CRC cells as well as the viability of CRC organoids in CTG assay.

Conclusion

Our results show that WNT11 is highly expressed in CRC and directly secreted by tumor cells. We show that WNT11 is a target gene of the Wnt pathway, which is upregulated in the context of APC mutations, and that WNT11 supports the proliferation of CRC cells. These results unravel new aspects of the oncogenic effects of APC mutations and identify WNT11 as a potential therapeutical target in APC-mutant CRC. To characterize the oncogenic function of WNT11 in CRC in more detail, further research is needed. The in vivo effects of WNT11 silencing will be addressed in a murine xenograft model.

Introduction

Intraductal Papillary Mucinous Neoplasias (IPMN) are cystic neoplasias of the ducts of the pancreas. They usually occur at advanced age and can progress to invasive adenocarcinomas. While there are morphological and spatial features to assess the progression risk, a mutational footprint defining the progression risk remains to be identified. IPMNs often harbor an activating mutation of either GNAS or KRAS or both, but neither interaction nor signaling crosstalk is well understood.

Aims & Methods

Here, we use a patient-derived induced pluripotent stem cell (iPSC) model system, harboring either a wildtype or mutant GNAS with an inducible oncogenic KRAS^G12D cassette. Various iPSC genotypes were differentiated towards the pancreatic lineage including pancreatic duct-like organoids, followed by subsequent transplantation onto the porcine urinary bladder (PUB) for long-term culture. Dysplasia, mucin expression and cyst size were subsequently analysed.

Results

Already 2 weeks post transplantation, a substantial amount of CK7/CK19 positive pancreatic ductal structures were found in all grafts with a strong cystic phenotype driven by a GNAS^R201C background. The phenotype was still present 8 weeks after transplantation. The induction of KRAS^G12D for 6 weeks resulted in an increased ductal lumen, which was further boosted by GNAS^R201C. In addition, a strong GNAS^R201C dependent expression of Mucin 2, a marker for the intestinal IPMN subtype was documented. Quantification of dysplastic ductal structures revealed an increase in low grade dysplasia driven either by KRAS^G12D or GNAS^R201C. Combination of both mutations was associated with a strong increase in the amount of high grade dysplastic ductal structures. In ductal structures a pro-proliferative phenotype driven by GNAS^R201C background was observed. The amount of p21 positive nuclei was highly increased by KRAS^G12D, while it was significantly attenuated in ductal cells with a double mutated KRAS^G12D GNAS^R201C background, showing a decreased amount of oncogene induced senescence. This was recapitulated in vitro in pancreatic ductal organoids.

Conclusion

The first results show an effect of GNAS^R201C on early dysplasia formation and a further boost of KRAS^G12D driven dysplasia and high-grade dysplasia formation. Additionally, the increased proliferative and decreased senescent phenotype hints at a GNAS^R201C driven bypass of oncogene induced senescence. Our genuine patient-derived iPSC model featuring an endogenous GNAS^R201C locus will allow us to closely investigate the early formation of IPMNs and timewise resolve their transition towards an invasive phenotype to further unravel the mechanisms of IPMN formation and the collaborative pro-oncogenic signaling of KRAS^G12D with GNAS^R201C.

Introduction

Many ulcerative colitis (UC) patients (pts) are suboptimally controlled on conventional therapies but are hesitant to move to biologics, indicating an unmet need for oral advanced therapies early in the treatment paradigm. Ozanimod (OZA), an oral, selective sphingosine 1-phosphate receptor modulator, is approved for the treatment of moderately to severely active UC in adults.

Aims & Methods

This post hoc analysis of the phase 3 True North clinical trial explored the efficacy of OZA in advanced therapy–naive UC pts who had active, symptomatic disease while on conventional therapies. OZA efficacy was assessed at Week (W) 10 in pts who had either moderate (Mayo endoscopy subscore [MES]=2) or severe (MES=3) endoscopic disease activity at baseline. Pts in Cohort 1 (C1) were randomized to OZA 0.92 mg or placebo (PBO) or received open-label OZA in Cohort 2 (C2) through W10. Pts were required to be on oral 5-aminosalicylate and/or corticosteroid for ≥2 wk before screening and continue through induction. Clinical endpoints (clinical remission, clinical response, endoscopic improvement, and mucosal healing) were assessed at W10. Treatment differences for OZA (C1) vs PBO were based on the Cochran-Mantel-Haenszel test, stratified by corticosteroid use at screening. Symptomatic clinical response and symptomatic clinical remission were assessed from W2 to W10.

Results

Of 616 advanced therapy–naive pts at baseline, 296 had moderate (PBO, n=64; OZA [C1], n=138; OZA [C2], n=94) and 320 had severe (PBO, n=73; OZA [C1], n=149; OZA [C2], n=98) endoscopic disease. Baseline pt characteristics were similar between groups, with higher prior immunomodulator use in severe (22%–34%) vs moderate (13%–23%) pts. Significantly higher symptomatic responses with OZA vs PBO were observed by W4 in moderate pts (OZA [C1] vs PBO: 67.4% vs 42.2%; difference: 23.0% [95% CI 8.6–37.3]) and by W5 in severe pts (OZA [C1] vs PBO: 51.0% vs 32.9%; difference: 17.6% [95% CI 4.1–31.2]). Similarly, significantly higher symptomatic remission rates were observed with OZA vs PBO by W5 in moderate pts (OZA [C1] vs PBO: 39.9% vs 18.8%; difference: 19.5% [95% CI 6.9–32.2]) and by W8 in severe pts (OZA [C1] vs PBO: 32.2% vs 16.4%; difference: 15.4% [95% CI 4.0–26.8]). OZA was more effective than PBO in achieving all clinical endpoints at W10 in moderate and severe pts (Table). Treatment differences (OZA [C1] vs PBO) were greater in moderate vs severe pts for clinical remission (22.8% [95% CI 11.7–33.9]; P<0.001 vs 8.0% [95% CI 1.7–14.2]; P=0.04), clinical response (27.2% [13.1–41.3]; P<0.001 vs 21.0% [8.2–33.8]; P=0.003), endoscopic improvement (28.7% [15.7–41.7]; P<0.001 vs 13.4% [5.2–21.7]; P=0.008), and mucosal healing (18.0% [8.1–27.9]; P=0.003 vs 5.4% [0.5–10.2]; P=0.09).

Conclusion

Pts with inadequate disease control while on conventional therapies were better controlled with OZA treatment than those who continued conventional treatments. Pts with moderate disease achieved greater benefit and responded more rapidly than those with severe disease. OZA is an effective oral advanced therapy option for pts suboptimally controlled on conventional therapies, especially those with moderate disease.

Disclosure

This study was funded by Bristol Myers Squibb, Princeton, NJ, USA.
PI: received lecture fees from AbbVie, Bristol Myers Squibb, Celgene, Celltrion, Dr. Falk Pharma, Ferring, Galapagos, Gilead, Janssen, MSD, Pfizer, Sandoz, Sapphire Medical, Shire, Takeda, Tillotts, and Warner Chilcott; received financial support for research from Celltrion, MSD, Pfizer, and Takeda; received advisory fees from AbbVie, Arena, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Celltrion, Eli Lilly, Genentech, Gilead, Hospira, Janssen, MSD, Pfizer, Pharmacosmos, Prometheus Biosciences, Roche, Samsung Bioepis, Sandoz, Takeda, TopiVert, VH2, Vifor, and Warner Chilcott.
MDL: received research funding from Pfizer and Takeda; consulted for AbbVie, Bristol Myers Squibb, Calibr, Eli Lilly, Genentech, Janssen, Pfizer, Prometheus, Roche, Salix, Takeda, Target RWE, and Theravance.
BS: consulted for AbbVie, Arena, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Falk Pharma, Galapagos, Janssen, Lilly, Pfizer, Prometheus, and Takeda; received speaker fees from AbbVie, CED Service GmbH, Falk Pharma, Ferring, Janssen, Novartis, Pfizer, and Takeda.
GD: served as an advisor for AbbVie, Agomab Therapeutics, Alimentiv, Applied Molecular Therapeutics, AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Celltrion, Cytoki, Eli Lilly, Exeliom Biosciences, Ferring, Galapagos, GlaxoSmithKline, Gossamer Bio, Immunic, Johnson & Johnson, Pfizer, Polpharma, ProciseDx, Progenity, Prometheus Biosciences, Prometheus Laboratories, Protagonist Therapeutics, Seres, Takeda, Tillotts, and Versant; and received speaker fees from AbbVie, Bristol Myers Squibb, Eli Lilly, Galapagos, Johnson & Johnson, Pfizer, Takeda, and Tillotts.
IB: received consulting fees from AbbVie, Amgen, Arena, Biogen, Bristol Myers Squibb, Celltrion, Ferring, Galapagos, Janssen, Pfizer, Shire, and Takeda; received speaker fees from AbbVie, Amgen, Arena, Biogen, Bristol Myers Squibb, Celgene, Celltrion, Ferring, Galapagos, Janssen, Mylan, Pfizer, Shire, and Takeda.
AJ, HW, and MTO: employees and/or shareholders of Bristol Myers Squibb.
DCW: received research funding from AbbVie, Bristol Myers Squibb, Genentech, Janssen, Pfizer/Arena, and Takeda; consulted for AbbVie, Bristol Myers Squibb, Eli Lilly, Janssen, Pfizer, and Takeda; received speaker fees from AbbVie, Bristol Myers Squibb, Janssen, and Takeda.
DTR: received grant support from Takeda; consulted for AbbVie, AltruBio, Aslan Pharmaceuticals, Athos Therapeutics, Bellatrix Pharmaceuticals, Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Chronicles, ClostraBio, Connect BioPharma, EcoR1, Eli Lilly, Genentech/Roche, Gilead Sciences, Iterative Health, Janssen, Kaleido Biosciences, Pfizer, Prometheus Biosciences, Reistone, Seres Therapeutics, Syneos, Takeda, Target RWE, and Trellus Health.