Ruozheng Wei

BACKGROUND AND AIMS: The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis. An imbalance in this highly regimented process within the intestinal crypts is associated with several intestinal pathologies. Although metabolic changes are known to play a pivotal role in cell proliferation and differentiation, how glycolysis contributes to intestinal epithelial homeostasis remains to be defined. METHODS: stem cells. Glycolysis was measured using the Seahorse XFe96 analyzer. Expression of phospho-p38 mitogen-activated protein kinase, the transcription factor atonal homolog 1, and intestinal cell differentiation markers lysozyme, mucin 2, and chromogranin A were determined by Western blot, quantitative real-time reverse transcription polymerase chain reaction, or immunofluorescence, and immunohistochemistry staining. RESULTS: HK2 is a target gene of Wnt signaling in intestinal epithelium. HK2 knockout or inhibition of glycolysis resulted in increased numbers of Paneth, goblet, and enteroendocrine cells and decreased intestinal stem cell self-renewal. Mechanistically, HK2 knockout resulted in activation of p38 mitogen-activated protein kinase and increased expression of ATOH1; inhibition of p38 mitogen-activated protein kinase signaling attenuated the phenotypes induced by HK2 knockout in intestinal organoids. HK2 knockout significantly decreased glycolysis and lactate production in intestinal organoids; supplementation of lactate or pyruvate reversed the phenotypes induced by HK2 knockout. CONCLUSIONS: Our results show that HK2 regulates intestinal stem cell self-renewal and differentiation through p38 mitogen-activated protein kinase/atonal homolog 1 signaling pathway. Our findings demonstrate an essential role for glycolysis in maintenance of intestinal stem cell function.

KRAS mutations serve as key oncogenic drivers in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). Despite the advancement of KRAS inhibitors, such as MRTX1133, for PDAC treatment, intrinsic and acquired resistance remain major barriers to their clinical efficacy. This study underscored the role of histone deacetylase 5 (HDAC5) loss in mediating intrinsic resistance to KRASG12D inhibitors. Mechanistically, HDAC5 promoted c-Myc degradation by deacetylating K148, thereby facilitating NEDD4-mediated ubiquitination at this site. The loss of HDAC5 resulted in hyperacetylation of c-Myc at K148, impeding the ubiquitination and subsequent degradation process of c-Myc following deacetylation. Consequently, c-Myc stability and transcriptional activity were sustained even under KRAS/MEK/ERK pathway inhibition, reinforcing MAPK signaling and promoting cell survival despite KRAS suppression. Our data further demonstrated that pharmacological or genetic inhibition of c-Myc effectively reversed the resistance phenotype mediated by HDAC5 loss, suggesting a therapeutic strategy centered on KRAS-MYC dual-node blockade. Furthermore, the expression levels of HDAC5 and the acetylation status of c-Myc may serve as biomarkers for predicting the therapeutic response to MRTX1133. These findings provide insights into overcoming resistance to KRASG12D inhibitors and offer potential biomarkers and combinatorial therapeutic strategies for precision treatment of PDAC.

BACKGROUND: ACK1, a non-receptor tyrosine kinase, phosphorylates various substrates involved in cancer progression. Its oncogenic activity is driven by gene amplification, mutations, and post-translational modifications. However, additional regulatory mechanisms that govern ACK1 activity remain to be fully understood. Liquid-liquid phase separation (LLPS) has emerged as a key mechanism of cellular compartmentalization, controlling the spatiotemporal dynamics of signaling pathways. METHODS: Expression plasmids and corresponding mutants were generated using molecular cloning techniques. Protein expression and localization were assessed through western blotting, immunofluorescence, and confocal microscopy. LLPS properties were evaluated using time-lapse imaging, photobleaching, optoDroplet assays, and in vitro droplet formation assays. Cellular functions were examined through colony formation and wound-healing assays. STAT5 signaling activation was assessed by western blotting, co-immunoprecipitation (Co-IP), immunofluorescence, RNA sequencing (RNA-Seq), and Gene Set Enrichment Analysis (GSEA). RESULTS: We demonstrate that ACK1 is frequently amplified and overexpressed in lung squamous cell carcinoma (LUSC). In LUSC cells, ACK1 undergoes LLPS, a process that depends on the intrinsically disordered region (IDR, 96-156 aa) but is independent of its kinase activity. We identify that the IDR induces droplet formation, with the 143-156 aa segment being essential for this activity. Furthermore, our data reveal that ACK1 phosphorylates STAT5 in LUSC cells. ACK1 condensates recruit the non-catalytic adaptors NCK1 and NCK2 and enhance STAT5 signaling. These condensates promote STAT5 nuclear localization and transcriptional activity, thereby facilitating LUSC cell growth and migration. CONCLUSIONS: Our findings highlight the crucial role of ACK1 condensates in oncogenic STAT5 signaling and suggest that targeting the formation of ACK1 condensates could serve as a potential therapeutic strategy for LUSC.