Introduction

Inflammatory Bowel Diseases (IBD) are gastrointestinal, auto-inflammatory disorders with a chronic relapsing and remitting course, mainly including Ulcerative Colitis (UC) and Crohn’s Disease (CD). They are multifactorial diseases which still lack a clear etiology, making identification of reliable predictors of disease course and therapy response of great interest to the medical community. The existence of extrachromosomal circular DNA fragments (eccDNA) is well established and has become a promising diagnostic and prognostic biomarker for different types of cancer. Herein, we investigate for the first time the role of eccDNA in patients affected by IBD.

Aims & Methods

Consecutive patients with an established diagnosis of IBD and non-IBD healthy controls who underwent colonoscopy for colorectal cancer screening were prospectively enrolled at Fondazione Policlinico Universitario “A: Gemelli” in this monocentric, observational, case-controlled study. In all IBD patients, disease activity was assessed using Partial Mayo Score (PMS) and Mayo Endoscopic Subscore (MES) for UC patients, and Harvey-Bradshaw Index (HBI) and Simple Endoscopic Score for CD (SES-CD) for CD. Colonic biopsies were collected from both inflamed and healthy mucosa of IBD patients and from non-IBD controls. Circular DNA was enriched, sequenced, and eccDNAs were identified with Circle Finder from intestinal biopsies. Circles containing the same gene were grouped together for downstream pathway analysis and defined as Produced per Gene eccDNAs (PpGC). Enrichr analysis was used to assess the pathways different PpGC belonged to.

Results

Forty IBD patients (19 with CD and 21 with UC) and 13 healthy controls (HC) were enrolled. Colonic mucosal samples from patients with IBD showed increased average eccDNAs than did intestinal samples from HC. This increase was observed across all chromosomes, indicating that a global increase in eccDNA production was across the human genome. Furthermore, there was a slight yet significant tendency for larger eccDNAs in IBD patients compared to HC.
Focusing only on eccDNA which came from genic elements, referred to as “genic eccDNAs”, approximately 60% of the functional DNA found on genic eccDNA in both groups were fragments from protein coding genes, 32% from non-coding RNAs and 8% from pseudogenes. Furthermore, even when only taking genic eccDNAs into account, IBD patients still had, on average, significantly more eccDNAs in the intestine when compared to non-IBD controls. Eighty PpGC were more abundant in IBD patients as compared to HC; enrich analysis revealed that pathways involved in cAMP signaling were significantly overrepresented in this gene set.
Considering only PpGCs which appear in at least 25% of patients, we found 157 PpGCs only found in UC patients and 210 PpGCs in CD patients, indicating that there may be some differences in eccDNA production between the two IBD diagnoses.
Moreover, on average, patients with IBD in remission had fewer eccDNAs in their intestines when compared to those with active disease, although the difference was not statistically significant. eccDNAs from the phosphodiesterase genes and genes involved in lipid metabolism were particularly enriched in patients with active UC and CD, compared to patients in remission.

Conclusion

We are the first to uncover an IBD-specific pattern of eccDNA production. Furthermore, we have identified genic hotspots which characterize active vs. inactive disease. We propose that eccDNA identification could be a promising new prognostic marker for IBD patients.

Disclosure

The authors declare no conflicts of interest.

Introduction

Gastric cancer (GC) is a multifactorial disease, with a significant microbial contribution. Beyond Helicobacter pylori, the bacterium most commonly linked to GC, lactic acid-producing bacteria (LAB) have been frequently reported to be more abundant in later stages of the gastric carcinogenesis cascade. LAB are known for producing considerably large amounts of the oncometabolite lactate, which is strongly linked to the main hallmarks of cancer (i.e., inflammation, immune escape, angiogenesis, invasion, and metastasis).

Aims & Methods

We aimed to understand the potential association of LAB with gastric precancerous lesions (GPL) and GC in two high-risk populations (Colombian Mestizo and Han Chinese). DNA and RNA were simultaneously extracted from snap frozen gastric biopsies from subjects presenting with functional dyspepsia, GPL (atrophic gastritis, intestinal metaplasia, and dysplasia), and GC. 16S gene (DNA, n=257) and transcript (cDNA, n=129) amplicons (V4 region) were sequenced with Illumina MiSeq 2x250 bp. Alpha, beta, and per-taxa analyses were performed.

Results

H. pylori significantly impacted the diversity and composition of the gastric microbiota. Gastric active microbiota (cDNA) assessment showed that there were significant differences in beta diversity between FD controls and GPL and GC patients. Further, there were strong positive associations between LAB, including Otu00023.Lactobacillus (100% similarity to Lactobacillus gasseri), Otu00045.Limosilactobacillus (100% similarity to Limosilactobacillus fermentum), Otu00046.Oribacterium (100% similarity to Oribacterium sinus), Otu00120.Streptococcus (100% similarity to Streptococcus anginosus), and Otu00232.Limosilactobacillus (100% similarity to Lactobacillus timonensis) and disease status (GPL and GC). These findings were maintained even when Otu00001.Helicobacter (100% similarity to H. pylori) was removed from the analysis, evidencing a strong signature of LAB regardless of the presence of H. pylori in the active gastric microbiota. Enrichment of Otu00046.Oribacterium and Otu00023.Lactobacillus in GPL and GC patients, was validated when the gastric resident/transient microbiota was assessed (DNA).

Conclusion

The gastric active microbiota of GPL and GC patients from two high-risk populations geographically and ethnically diverse is significantly dominated by LAB (Otu00046.Oribacterium and Otu00023.Lactobacillus), demonstrating a potential role for these bacteria in the pathogenesis of GC. Assessment of the viable gastric microbiota appears to be more informative compared to resident/transient microbiota assessment.

Introduction

Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome caused by mutations in the APC gene, a negative regulator of the Wnt signaling pathway. It is characterized by the development of hundreds to thousands of Wnt-driven adenomatous polyps in the colon. To prevent the inevitable progression to colorectal cancer, patients with FAP typically undergo total proctocolectomy in early adulthood. Currently, no effective medical therapy exists for FAP. Casein kinase I alpha (CKIα), another negative regulator of Wnt signaling, has emerged as a potential therapeutic target. Prior work in our lab demonstrated that, unlike APC loss, epithelial deletion of Csnk1a1 (CKIα) in the gut does not induce tumorigenesis. Instead, it activates a non-canonical DNA damage response, leading to robust p53 stabilization and suppression of tumorigenic changes. Given that TP53 mutations are typically late events in the adenoma–carcinoma sequence, early-stage lesions in FAP often retain wild-type p53. Unlike sporadic adenomas, which can be resected during colonoscopy, the high polyp burden in FAP precludes endoscopic management, underscoring the need for effective chemoprevention.

Aims & Methods

Our aim in this work was to investigated CKIα as a therapeutic target in preclinical models of FAP.
We employed Csnk1a1^fl/fl, APC^fl/fl, Villin-CreERT2, and CDX2-CreERT2 transgenic mice for in vivo studies, with tamoxifen-inducible gene ablation. Intestinal organoids derived from normal and tumor tissues were used for ex vivo experiments.

Results

In vivo, simultaneous deletion of APC and CKIα in intestinal epithelium did not further enhance Wnt target gene expression compared to APC loss alone. Instead, dual knockout (DKO) stabilized p53, promoted apoptosis, and blocked the dysplastic progression normally observed in APC-deficient gut tissue. In organoid models, treatment with the novel CKIα degrader BTX1050 selectively inhibited tumor-derived organoid proliferation, but did not fully eliminate tumor-initiating cells. We therefore tested A51, a dual CKIα and CDK7/9 inhibitor developed in our lab, which both stabilized and activated p53 and suppressed transcription of super-enhancer-driven oncogenes. A51 treatment significantly downregulated Myc and Mcl1, increased apoptosis in tumor organoids, but only partially suppressed tumor growth in vivo. Notably, A51 induced MAPK pathway activation, as indicated by increased pERK levels. This prompted combination therapy with A51 and the MEK inhibitor trametinib, which demonstrated potent synergistic anti-tumor activity in both organoid and mouse models.

Conclusion

CKIα depletion exerts anti-tumor effects in intestinal tumor models with wild-type p53. Our findings support the combination of A51 and MEK inhibition as a promising "chemical polypectomy" approach for FAP patients.

Disclosure

n/a