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

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

Src-family tyrosine kinases (SFKs) are mutated or aberrantly regulated in up to 80% of colorectal cancers and are central drivers of oncogenic signaling. Therapeutically targeting SFKs has proven difficult due to compensatory signaling and mechanisms that stabilize these proteins. Their membrane localization and stability are normally governed by N-terminal myristoylation, a co-translational modification of Gly2 (glycine) following initiator methionine removal. Loss of this modification disrupts subcellular localization and exposes Gly2 to N-degron pathways, which mediate proteasomal degradation based on the identity and modification state of the N-terminus.

Aims & Methods

We aimed to define how omission of N-terminal myristoylation affects SFK stability and degradation, and to identify the responsible E3 ligases. To this end, we employed peptide pulldowns combined with mass spectrometry, in vitro binding assays, and AlphaFold3-based interaction predictions. Functional validation included knockdown of NMT1/2 and DCAF10, CRISPR/Cas9-mediated knockout of LYN, and re-expression of N-terminal Lyn mutants. In vitro ubiquitination assays with purified DCAF10-DDB1-CUL4A complex confirmed E3 ligase activity toward acetylated Gly substrates.

Results

Loss of myristoylation led to increased turnover of Src-family kinases. Free N-terminal Gly residues were recognized by the known CUL2-ZYG11B/ZER1 complex. Unexpectedly, we also observed aberrant N-terminal acetylation of Gly2, which triggered degradation via the DCAF10-DDB1-CUL4A E3 ligase complex. Knockdown of DCAF10 stabilized acetylated Gly–Lyn in cells, and in vitro assays confirmed direct recognition and ubiquitination of this substrate. Both pathways acted in parallel, ensuring efficient clearance of non-myristoylated Lyn variants.

Conclusion

Our findings identify a dual N-degron surveillance mechanism targeting non-myristoylated SFKs: CUL2-ZYG11B/ZER1 acts on free Gly residues, while DCAF10-DDB1-CUL4A targets aberrantly acetylated Gly. This quality control system prevents the accumulation of mislocalized or dysfunctional SFKs. Beyond degradation, N-terminal modifications also regulate localization and signaling: while myristoylation anchors SFKs at membranes, non-myristoylated or G2A-mutant forms—typically N-terminally acetylated—accumulate in the nucleus when export is blocked, suggesting that myristoylation actively restricts nuclear localization. Given emerging evidence for nuclear SFK functions, our data suggest that alternative N-terminal modifications and their recognition by distinct degradation pathways fine-tune SFK stability, localization, and oncogenic output—highlighting new opportunities for targeted cancer therapies.

References

Timms, R. T. et al. A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation. Science 365, (2019).

Disclosure

We declare no conflict of interest