Loss of polarity by Cdc42 depletion and oncogenic Kras activation in the mouse intestinal epithelia leads to a necrotizing enterocolitis (NEC)-like disease

Domain: biology
Published: 2026-03-18
Editorial outcome: major_revision

Abstract
Cell polarity is essential for maintaining intestinal epithelial organization and function. Here we show that combined loss of polarity by epithelial loss of Cdc42 with oncogenic Kras expression in mice causes small intestine failure leading to weight loss, inflammation, epithelial necroptosis, and lethality. These phenotypic defects are characterized by a loss of intestinal stem cells, disrupted epithelial architecture, altered hippo signaling, elevated inflammatory cytokines, and activation of necroptotic cell death, that closely resemble necrotizing enterocolitis (NEC). Single-cell transcriptomic analysis reveals a coordinated dysregulation of polarity machinery, inflammatory pathways, and necroptosis program. Suppression of YAP, IL-1, TNFα signaling or necroptosis rescues the intestinal pathology. Similar NEC-like phenotypes arise when Cdc42 loss and oncogenic Kras activation are initiated from intestinal stem cells. These findings provide mechanism insights involving polarity-YAP-IL1/TNFα signaling induced necroptosis for the synergistic effect of hyperactivation of SKras signaling and loss of polarity in disrupting intestinal epithelia.

Introduction
Cdc42 is a member of the Rho GTPase family widely recognized for its role in regulating actin cytoskeleton dynamics, adhesion/cell junction signaling, and polarity 1-3. In the intestine, Cdc42 is particularly important for maintaining epithelia polarity, which is essential for the formation and function of the crypt-villus axis, as loss of Cdc42 in intestinal epithelial cells leads to a broad tissue hyperplasia accompanied by hyperproliferation of transient amplifying (TA) cells and intestinal stem cells (ISCs), crypt enlargement, and defective Paneth cell differentiation and localization in the intestine 4. Recent studies further highlighted a role for Cdc42 in regulating the transition of intestinal stem cells to transit-amplifying cells, implicating it in intestinal homeostasis and ISC differentiation 5. Interestingly, despite dramatic hyperproliferation of the Cdc42-null crypt cells due to upregulated hippo signaling, intestinal epithelia Cdc42 knockout (KO) mice do not develop tumors and have a mostly normal life expectancy 4.

Oncogenic KRAS mutations are commonly found in colorectal cancer (CRC) resulting in constitutive activation of the Ras signaling pathway, and is causal for uncontrolled cell proliferation and survival 6-8. The KRAS G12D mutation has been shown to drive the development of CRC by activating downstream signaling pathways including RAF/MEK/ERK to promote unchecked cell growth and resistance to apoptosis 9,10. While the KRAS mutation is pivotal in the progression and maintenance of CRC, they act in conjunction with additional genetic and epigenetic alterations, and the progression to CRC requires further genetic mutations 11,12.

Since loss of polarity is a hallmark of intestinal cancer, we are interested in investigating the combined effect of Cdc42 loss and oncogenic Kras G12D in the intestine epithelia, as either mutation can induce hyperplasia and the hyperproliferation induced by Kras G12D and loss of polarity caused by Cdc42 KO might synergize to drive a transformation to carcinoma. To better understand how the combined loss of polarity, a known phenotype in epithelial cancers, with oncogenic Kras mutation, we have generated a genetic mouse model with simultaneous inducible Cdc42 deletion and oncogenic Kras G12D activation. Surprisingly, these mice developed necrotizing enterocolitis (NEC)-like defects, which is one of the most common and devastating gastrointestinal conditions primarily affecting premature infants characterized by inflammation and necrosis of the bowel 13,14, leading to lethality of the mice within a short time frame. Corroborating with this observation, an analysis of human colon adenocarcinoma datasets from The Cancer Genome Atlas (TCGA) database reveals that the expression of KRAS mutants and defects in polarity-related molecules including Cdc42, Dlg5, and Scrib are mutually exclusive in colon cancer patients. We further identify elevated YAP signaling and IL-1/TNFα mediated inflammation and necroptosis in the mouse model as key mediators of the development of NEC-like defects. Our study highlights a interaction between Cdc42 loss and oncogenic Kras G12D in the intestinal epithelium, which, instead of promoting proliferation and transformation, results in a severe NEC-like condition.

Results
Cdc42 deletion combined with oncogenic Kras G12D mutation in intestinal epithelium disrupts epithelial morphology and causes lethality

To generate a mouse model to study the combined effects of Cdc42 KO and oncogenic Kras mutation in the intestine, intestinal epithelium-specific inducible villin-CreERT2 (villin-CreER) expressing mice were crossbred with Cdc42 flox/flox mice 4, and with the KrasLSL-G12D mice to generate villin-CreER; Cdc42flox/+(hereafter referred to as WT) mice, villin-CreER; Cdc42flox/flox (hereafter referred to as Cdc42 KO) mice, villin-CreER; KrasLSL-G12D (hereafter referred to as Kras) mice, and villin-CreER; Cdc42flox/flox; KrasLSL-G12D + (hereafter referred to as Cdc42 KO/Kras) mice. The Cdc42 KO/Kras mice became severely ill after tamoxifen (TAM) injections to run on the mutations. We analyzed the morphology of the Cdc42 KO/Kras mice and found a severely disrupted intestinal epithelium in both the small intestine and colon that showed broken villi and tissue hypoplasia (Figure 1A, Supplemental Figure 1). Meanwhile, Cdc42 KO mice only showed disrupted polarity in the villi, consistent with previous findings 5, while Kras mice exhibited no noticeable differences in the intestine epithelia compared to that of WT mice (Figure 1A). The Cdc42 KO/Kras mice rapidly lost 15% of their body weight within 96 hours after TAM injections, and 100% of them did not survive beyond 7 days. In contrast, Cdc42 KO or Kras mice showed comparable body weight and survival rate to WT mice (Figures 1B, C). Western blot analyses of isolated intestinal epithelia confirmed the loss of Cdc42 protein in the intestines of Cdc42 KO and Cdc42 KO/Kras mice, and the activation of Kras downstream targets p-ERK and p-AKT (Thr308) in the intestines of Kras and Cdc42 KO/Kras mice were apparent (Figure 1D). To examine the cause of lethality of Cdc42 KO/Kras mice, we performed a TUNEL assay to analyze cell death in the intestine and found a significantly elevated TUNEL+ cell death in the villi area of the Cdc42 KO/Kras small intestine that were absent in mice of the other genotypes (Figure 1E, F, Supplemental Figure 1). Co-staining with a nutrient-absorptive enterocyte marker ALPi suggests that enterocytes are the main cell type in the intestine dying in the Cdc42 KO/Kras mice, which could explain the rapid loss of body weight and subsequent lethality of the mice (Figure 1E). The expression of the tight junction marker ZO-1 is also altered in Cdc42 KO/Kras mice, indicating disrupted tight junctions (Figure 1E).

Cdc42 KO/Kras G12D double mutations cause reduced ISCs, mis-localized Paneth cells and disrupted basal-lateral polarity

To examine the changes of the major cell types in the Cdc42 KO/Kras intestine, we performed immunostaining of multiple cell type specific markers in the fixed duodenum samples. The percentage of epithelial cells expressing the enterocyte marker ALPi in Cdc42 KO/Kras mice was reduced by over 60%, while the epithelial cells became round and dissociated, suggesting a loss of epithelial function (Figures 2A, G). The number as well as the mucin secretion related genes in goblet cells and hormone expression related genes in enteroendocrine cells did not significantly change in the Cdc42 KO/Kras mice (Figures 2B, C, Supplemental Figure 2A, B). However, the localization of Paneth cells in CDdc42 KO/Kras mice was altered, with Paneth cells found in the villi area versus the crypt area, a pattern seen in the Cdc42 KO mice, whereas all Paneth cells reside at the bottom of the crypts in the WT mice (Figures 2D, H). Similar to Cdc42 KO, the ISC population in Cdc42 KO/Kras crypts reduced significantly (Figure 2E, I), and basolateral polarity was disrupted in the Cdc42 KO/Kras villi (Figure 2F, Supplemental Figure 2C). mRNA expression of the ISC marker Lgr5, the Paneth cell marker Defa5, and the proliferation marker Cyclin D1 were also reduced in the Cdc42 KO/Kras intestine, suggesting an impaired ISC and its niche functions (Figure 2J).

Single cell RNAseq analyses reveal elevated inflammation, necroptosis, and disrupted adhesion junction in Cdc42 KO/Kras G12D mutant intestine

To explore the underlying mechanism of the disrupted morphology and the loss of enterocytes and ISCs in Cdc42 KO/Kras mice, we isolated villi/crypt cells from WT, Cdc42 KO, Kras, and Cdc42 KO/Kras mice and performed high-throughput single-cell mRNA-sequencing (scRNAseq) and uniform manifold approximation and projection (UMAP) clustering analysis, a nonlinear dimensionality-reduction technique 23. Unbiased clustering of isolated villi/crypt cells from all four samples, followed by cluster annotation based on cell marker expressions 5 revealed at least seven intestinal cell types: Enteroendocrine cells, enterocytes, Goblet cells, Panet cells, ISCs, transit-amplifying (TA) cells, and tuft cells, as well as major immune cell types: macrophages, B cells, and T cells (Supplemental Figure 3A-C). We performed gene set enrichment analysis (GSEA) and found that acute inflammation, necroptosis, and response to wound pathways were significantly elevated in the combined gut cell populations (enterocytes, enteroendocrine cells, Goblet cells, Paneth cells, ISCs, TA cells, Tuft cells) in the Cdc42 KO/Kras mice, whereas cellular respiration pathway signature genes were reduced (Figure 3A). Similar pathway changes were observed in the three gut cell subtypes, i.e. enterocytes, ISCs/TA cells and Goblet cells, when analyzed individually (Supplemental Figure 3C). This suggests that the Cdc42 KO/Kras mice are experiencing an acute inflammatory response and necroptosis-mediated cell death. Adhesion junction pathways and actin filament pathways are also altered in the Cdc42 KO/Kras intestine (Figure 3A). Three major necroptosis pathway marker genes: tnfrsf23, bok, tnf were enriched in the enterocyte cluster in the Cdc42 KO/Kras genotype (Figure 3B, Supplemental Figure 3B), which is consistent with the TUNEL staining pattern in the villi. In addition, we found that YAP signaling, a known regulator of ISC regeneration upon injury 24,25 and a critical mediator of the ISC-to-TA cell fate transition 5, is elevated in Cdc42 KO and Cdc42 KO/Kras TA cells/ISCs (Figure 3C), suggesting a potential role in the observed inflammation and necroptosis/cell death phenotypes.

Cdc42 KO/Kras G12D mutant shows reduced proliferation and increased necroptosis and inflammation in intestine

To verify the relevance of the scRNAseq analyses, particularly related to the inflammatory response and necroptosis changes in the Cdc42 KO/Kras intestine, we first examined proliferation using Ki67 and phospho-histone H3 staining. We found that crypt cell proliferation was significantly elevated in the Cdc42 KO and Kras mice compared to WT, but was tempered in Cdc42 KO/Kras mice (Figure 4A, B, G). Apoptosis, marked by cleaved caspase-3, was not evident in the Cdc42 KO/Kras mice (Figure 4C), suggesting that the observed cell death is not due to programmed cell death. Caspase-1 plays a critical role in the inflammatory response by mediating the activation of pro-inflammatory cytokines, particularly interleukin-1β (IL-1β) 26,27. We found that caspase-1 is enriched in the entire epithelium of the Cdc42 KO/Kras duodenum, but less so in the distal small intestine (ileum) or colon (Figure 4D, H, Supplemental Figure 1). Additionally, interleukin-1β (IL-1β) and tumor necrosis factor (tnf), two other key pro-inflammatory cytokines that play critical roles in the inflammatory response, are also elevated in the Cdc42 KO/Kras mice (Figure 4I). Necroptosis markers pMLKL and RIP 28,29 are overexpressed in the Cdc42 KO/Kras epithelium in small intestine and colon (Figure 4E, F, H, Supplemental Figure 1), consistent with the scRNA-seq results. Immunofluorescence staining for macrophages, T cells, and TNFα shows their accumulation primarily in the top region of the villi, including capillaries and lacteals (Supplemental Figure 4A), while flow cytometry analysis reveals that multiple immune cell populations are increased in the Cdc42 KO/Kras mice, including macrophages, neutrophils, T cells, and B cells (Figure 4J, Supplemental Figure 4B). These findings indicate that necroptosis and an elevated immune response, characterized by increased immune cells accumulation and overexpression of inflammatory cytokines and necroptosis markers, contribute to the severe deterioration of the intestine epithelia of the Cdc42 KO/Kras mice.

Inhibition of necroptosis or IL1/TNFα-mediated inflammation rescues defects of Cdc42 KO/Kras G12D mutant intestine

The defects observed in the Cdc42 KO/Kras mice, i.e. weight reduction, disrupted villous mucosal structure, altered expression and localization of tight junction proteins, inflammatory response, and necrosis, are typical characteristics of the NEC mouse models 30. To date, the pathogenesis of NEC is not entirely understood, but recent studies indicate that it is multifactorial involving the immaturity of the intestinal barrier system, ischemic injury to the intestine, and hyperinflammation 13. To understand whether necroptosis (and injury to the intestine) and/or hyperinflammation is a leading cause of NEC-like defects in the Cdc42 KO/Kras intestine, we performed a “rescue” experiment using the necroptosis inhibitor Nec-1s 31 or the IL-1R inhibitor anakinra 32,33.

Administration of the necroptosis inhibitor Nec-1s to Cdc42 KO/Kras mice improved the general morphology of the duodenum and restores the integrity of the villous mucosal structure (Figure 5A). pMLKL staining along with reduced TUNEL signals confirmed the efficacy of necroptosis inhibition in the Cdc42 KO/Kras epithelium (Figure 5B, G, H), suggesting that the inhibition of necroptosis is sufficient to prevent the intestine cell death phenotype in the Cdc42 KO/Kras mice. Consistently, the Cdc42 KO/Kras mice treated with Nec-1s showed reduced body weight loss and exhibited improved survivability (Figure 5I, Supplemental Figure 5A). Interestingly, the populations of ISC and proliferative cells in the crypts were also restored by Nec-1s (Figure 5D), suggesting that necroptosis in the villi impacted the TA cell/ISC populations in the crypts. To further assess the ISC function, we performed an enteroid colony growth assay using Nec-1s–rescued crypts. While Nec-1s treatment restored ISC survival and allowed formation of enteroid spheres compared to untreated Cdc42 KO/Kras ISCs, their clonogenic potential was not fully rescued (Supplemental Figure 5D), indicating that the rescue of ISC number and survival is incomplete and that additional functional defects persist beyond cell survival. Not surprisingly, the necroptosis inhibitor restored the enterocyte population in the Cdc42 KO/Kras epithelium (Supplemental Figure 5B) whereas the mis-localization of Paneth cells and loss of basolateral polarity were not rescued (Figure 5E, Supplemental Figure 5B). In addition, macrophages and neutrophils were reduced but caspase-1 remained overexpressed by Nec-1s (Figure 5C, F, H, J, Supplemental Figure 5C).

To further explore other forms of programmed cell death such as pyroptosis, we examined cleaved gasdermin D and found that it was upregulated in Cdc42 KO/Kras mice but was not rescued by necroptosis inhibition (Supplemental Figure 5B), indicating an activation of the pyroptosis pathway in the double mutant mice. This observation is consistent with the finding that although inhibition of necroptosis reduced epithelial cell death and extended survival (Figure 5I), approximately 60% of Nec-1s–treated mice still exhibited a delayed mortality, suggesting that unresolved pyroptosis may also contribute to lethality in Cdc42 KO/Kras mice.

Similar to Nec-1s, intraperitoneal injection of the IL-1R inhibitor anakinra or anti-TNFα antibody also restored the morphology of Cdc42 KO/Kras mice (Figure 6A, Supplemental Figure 6C) and reduced necroptosis, inflammation and cell death in the villi (Figure 6B, G, H, Supplemental Figure 6A-C), implicating IL-1/TNFα pathway in the overall phenotypes of the double mutants and suggesting a positive feed forward loop between IL-1/ TNFα-mediated hyperinflammation and necroptosis. Notably, IL-1R inhibition effectively suppressed immune cell–derived IL-1β and TNFα expression (Supplemental Figure 6A), indicating that the inflammatory signaling and necroptosis phenotypes are largely driven by inflammation-derived microenvironmental cues. In contrast, epithelial-intrinsic defects, such as disrupted polarity marked by Par3 and Na⁺/K⁺-ATPase disorganization (Figure 6D, Supplemental Figure 6A) and altered epithelial morphology (Figure 6A), were not restored, suggesting that disrupted adhesion junctions and polarity pathways are intrinsic to the epithelium. Enterocytes, ISCs, and proliferating cells were also restored by the IL-1 inhibitor treatment (Figure 6C, E, F, I, J), while the localization of Paneth cells and basolateral polarity were not rescued (Figure 6D, E, Supplemental Figure 6B). Given the critical niche-supporting role of Paneth cells for ISCs, we considered whether their mislocalization might contribute to ISC depletion. Previous studies have demonstrated that Paneth cells are dispensable for ISC maintenance and function 34. Consistent with this, Paneth cell mislocalization alone is not sufficient to cause ISC loss in our models, as ISC recovery was observed in the above rescue experiments with Nec-1s or IL1R inhibitor, despite persistent Paneth cell mislocalization.

These results from the administration of necroptosis or IL-1R inhibitors in Cdc42 KO/Kras mice demonstrate a significant restoration of intestinal morphology and proliferation, highlighting the interplay between necroptosis and hyperinflammation in the pathogenesis of the NEC-like defects. Despite improvements in enterocyte and ISC populations, mis-localization of Paneth cells and loss of basolateral polarity remained unaffected, indicating that necroptosis or inflammation is likely downstream of epithelium loss of polarity and intestinal disruption.

ISC-specific Cdc42 KO/Kras G12D mutations cause similar NEC-like defects as intestinal epithelia-targeted mutations

We observed a loss of stemness marker Olmf4 in the Cdc42 KO/Kras mice, which can be restored by inhibition of necroptosis and inflammation. To investigate the possible effect of Cdc42 KO and Kras activation from ISCs leading to the NEC-like phenotypes, we utilized an inducible ISC-specific Cdc42 KO/Kras line. This was achieved by breeding Cdc42 flox/flox mice with Olfactomedin-4 (Olfm4)-CreER knock-in mice 15 to generate Olfm4-CreER; Cdc42flox/+(hereafter referred to as O-WT) mice, Olfm4-CreER; Cdc42flox/flox (hereafter referred to as O-Cdc42 KO) mice, Olfm4-CreER; KrasLSL-G12D +( hereafter referred to as O-Kras) mice, and Olfm4-CreER; Cdc42flox/flox; KrasLSL-G12D + (hereafter referred to as O-Cdc42 KO/Kras) mice. Following TAM injection, the O-Cdc42 KO/Kras mice experienced weight loss and lethality as observed in the villin-CreER driven Cdc42 KO/Kras mutant strain (Figure 7I), albeit in a slower time course. We examined the O-Cdc42 KO/Kras mice five days after TAM injection and observed similar defects as those seen in the villin-CreER Cdc42 KO/Kras mice, including disrupted epithelium, increased cell death in the villi, loss of polarity, and loss of ISC stemness marker (Figure 7A-D). These results suggest that loss of Cdc42 combined with Kras activation in ISCs is sufficient to initiate NEC-like defects as the mutant ISCs can differentiate into mature intestinal tissues to propagate defects to the enterocytes in the villi.

Since YAP signaling mostly resides in the crypt ISC /TA cells and is elevated in the Cdc42 KO/Kras intestine (Figure 3C), we further examined whether the increased YAP activity plays a role in the development of NEC-like defects. Intraperitoneal administration of the YAP inhibitor Verteporfin to O-Cdc42 KO/Kras mice was able to rescue the general morphology of the intestinal epithelium (Figure 7E) and reduce cell death, necroptosis, and inflammatory response of the mutant (Figure 7F, J, K, Supplemental Figure 7A). YAP inhibition also restored crypt cell proliferation (Figure 7G, L) and ISC marker expression (Figure 7H, M), though it did not correct the mislocalization of Paneth cells (Figure 7G). Flow cytometry analysis revealed that the macrophages in O-Cdc42 KO/Kras mice were reduced (Supplemental Figure 7B). The nuclear YAP expression was not impacted by Nec-1s or IL1R inhibitor, suggesting that elevated YAP signaling is upstream of the necroptosis and IL1/TNFα-mediated inflammatory feed forward loop (Supplemental Figure 7C). These results indicate that elevated YAP signaling in the ISC/TA crypt cells contributes to the Cdc42 KO/Kras phonotypes in the villi. They provide insights into the mechanism underlying the intestinal tissue damage - elevated YAP signaling by Cdc42 KO aided by a strong oncogenic cue such as Kras G12D mutation from the crypt progenitor cells can induce inflammation and necroptosis, leading to NEC-like defects.

Methods
Animal Studies

All experiments were performed in accordance to guidelines from the Institutional Animal Care and Use Committee (IACUC) at Cincinnati Children’s Hospital Medical Center (protocol # IACUC2023-0016). We used a Cdc42flox/flox mouse line based on previous validations 4. KrasLSL-G12D mice were purchased from Jackson Lab (RRID:IMSR_JAX:008179) and were based on manufacturer's validations. The Cdc42flox/flox mouse and/or KrasLSL-G12D mice was crossed with Villin-CreERT2 mice provided by Dr. Helen Piwnica-Worms at the University of Texas MD Anderson Center, or Olfm4-IRES-eGFPCreERT2 mice 15 provided by Dr. Hans Clevers at University of Utrecht. Littermate controls were generated by standard pairings. Animals are housed in an AALAC accredited barrier facility of CCHMC that meets or exceeds the Animal Welfare Act requirements under a light cycle – on from 6am-8pm, temperature set point at 72 degrees, humidity range of 30%-70%. Euthanasia was performed by CO2 followed by cervical dislocation. To induce Cre recombinase, 2-3 months old adult mice (mixed gender, no phenotypical difference between male or female mice) were intraperitoneally injected with 2 mg of tamoxifen (Sigma-Aldrich, Switzerland) dissolved in corn oil for 3 consecutive days from Day 1 to Day 3. Mice were sacrificed and analyzed 5 days after the first injection (Day 5). Nec-1s (5mg/kg body weight per day), IL-1R inhibitor (anakinra, 50mg/kg body weight per day), anti-TNFα antibody (2.5mg/kg body weight, once, on day 0) and YAP inhibitor (Verteporfin, 50mg/kg body weight per day) were intraperitoneally injected for 5 consecutive days from Day from Day 0 to Day 4.

H&E staining and tissue preparation

In addition to the above-described mouse tissues, the study used NEC and normal donors’ small intestine tissue samples from the Discover Together Biobank at Cincinnati Children’s Research Foundation. The intestinal tissues were flushed with PBS and fixed in 10% formalin or 4% paraformaldehyde overnight at 4 degree Celsius. Tissues were then embedded in paraffin or cold optimum cutting temperature embedding medium. Processing of tissues for paraffin-embedding and H&E staining was performed by the CCHMC Digestive Health Center Pathology Core.

Immunofluorescence and Confocal microscopy

Immunofluorescence was performed on frozen sections. Intestinal tissues were flushed with PBS and fixed in 4% paraformaldehyde overnight at 4°C, cryoprotected in 30% sucrose, embedded in OCT compound, and sectioned at 12 μm. Antibodies for immunofluorescence are in Supplementary Data 1 with 1:250 dilution for primary antibody and 1:800 dilution for secondary antibody. Images were acquired on a Nikon A1R GaAsP Inverted Confocal Microscope, with signal intensity optimized for control samples and the same settings (laser power/gain/offset) used for all tissue for the same experiment.

Villus/Crypt Isolation

Sections of intestine (duodenum, ileum, and colon; 5–6 cm in length) were harvested, flushed with ice-cold phosphate-buffered saline (PBS), opened longitudinally, and immersed in fresh ice-cold PBS. The tissue was then cut into five equal pieces and transferred to 10 ml PBS containing 10 mM EDTA. The tube was shaken vigorously for 20 s, and the supernatant containing debris was discarded before adding fresh PBS–EDTA. The samples were incubated for 30 min at 4 °C, with vigorous shaking for 20 s every 10 min. After incubation, the intestinal pieces were removed, and villus/crypt epithelial cells together with immune cells were collected from the PBS–EDTA solution.

Western blotting

After intestinal epithelium from villi/crypts was isolated, tissues were then homogenized in lysis buffer containing protease and phosphatase inhibitors, sonicated at 4°C, mixed with 4× sodium dodecyl sulfate loading buffer and heated at 100°C for 5 minutes. A Bradford assay was used to determine protein concentration (Bio-Rad). Antibodies used for Western blots are in the Supplementary Data 1 with 1:400 dilution for primary antibody and 1:800 dilution for secondary antibody. Uncropped and unprocessed scans of the most important blots are in the Source Data file.

RNA extraction and Quantitative Real-Time PCR

mRNA was isolated from intestinal crypts using an RNA mini kit (Qiagen). RNA concentration was measured by a Nanodrop and cDNA was produced with a cDNA synthesis kit (ABI). Quantitative PCR was performed using Taqman qPCR master mix (ABI) on a Bio-rad CFX Opus 96 quantitative reverse-transcription polymerase chain reaction (qRT-PCR) instrument. Relative expression levels were determined by the ΔΔCt method standardized to beta-actin.

Quantification

phospho-histone H3 (pH3)/Ki67/TUNEL positive cell number was quantified by counting the average number of pH3/Ki67/TUNEL positive cells in one villus and one adjacent crypt (10 villus/crypt per mouse). ALPi+ cell percentage was quantified by counting the number of ALPi positive epithelial cells in one villus, and then divided by the total number of epithelial cells in the name villus (10 villus per mouse). Relative intensity of Olfm4/Caspase-1/pMLKL was quantified by measuring the YAP immunostaining intensity using ImageJ (Version 2.14.0), which is normalized to Dapi nuclei staining (10 villus/crypt per mouse).

Statistics

Results are expressed as the mean ± standard deviation. Significance was calculated by Student’s t-test. P<0.05 was considered significant. Sample size was ≥ 3 for statistical analysis. They are chosen based on feasible animal cohorts and sufficient statistic power considerations.

Single-cell suspension preparation

After intestinal epithelium from villi/crypts was isolated, villi/crypts were resuspended in mixed medium comprised of 30% 10x TrypLE and 70% Advanced DMEM/F12 , incubated at 37 degree for 30-45 min. Mixing and passaging through a glass pipette was preformed every 10 min and suspensions were monitored with a microscope. After disruption of the cell aggregates, cells were filtered through a 40 mm filter and then pelleted by centrifugation (5 min at 300–400g), washed once with Advanced DMEM/F12, and resuspended in the same medium for 10x genomics sc-RNA seq.

Flow Cytometry

After single-cell suspensions were prepared, flow cytometry was performed to analyze neutrophils and macrophages based on surface marker expression. Cells were resuspended in staining buffer (PBS containing 2% fetal bovine serum) and incubated with fluorochrome-conjugated antibodies (purchased from BD Biosciences) at 4 °C for 30 minutes. The following antibodies were used: CD45-FITC (553080), CD11b-APC/Cy7 (557657), Gr-1-BV510 (563040), and F4/80-PE (565410). After staining, cells were washed with staining buffer, and DAPI (Thermo Fisher Scientific, D1306) was used to exclude dead cells. Samples were acquired on a BD LSRFortessa flow cytometer, and data were analyzed with BD FACSDiva or FlowJo software. Neutrophils were defined as CD45⁺CD11b⁺Gr-1⁺ cells, and macrophages as CD45⁺F4/80⁺ cells.

RNA Library Prep and Sequencing

After single cell suspension, a 3ʹ Gene Expression Library was prepared for sc-RNA seq by the CCHMC DNA Genotyping and Sequencing Core, using “Chromium Single Cell 3ʹ Reagent Kits 3v3.1” by 10x Genomics. Protocol by 10x Genomics in CG000183 Rev B user guide was strictly followed during the construction of the library, and 14 cycles were run during section 2.2 cDNA amplification. The NovaSeq 6000/ NovaSeq X Plus Sequencer was used with the sequencing read parameters designated in the user guide.

Single-cell data processing

Samples were sequenced on the Novaseq 6000 platform (RRID:SCR_016387). Resulting fastqs were processed through the CellRanger pipeline v5.0.1 (RRID:SCR_017344) using 10X Genomics’ mm10-2020-A reference genome, with the setting include introns set to false. Background reads were removed using decontX from the celda package using the filtered barcodes as the cells to keep and the remaining cells in the raw barcode matrix as the background 18. Doublets were minimized using DoubletFinder (RRID:SCR_018771) 19. The R v4.4 (RRID:SCR_001905) library Seurat v4.4 (RRID:SCR_016341) 20 was used for cell type clustering and marker gene identification. Cells expressing >500 genes were retained for downstream analysis. Each sample was normalized SCTransform v2 (RRID:SCR_022146), using the glmGamPoi method and the number of RNA molecules per cell were regressed out. Samples were integrated with common anchor genes using the rPCA method to minimize sample to sample variation. Cell clusters were determined by the Louvain algorithm using a resolution of 0.2. UMAP dimension reduction was done using the first thirty principal components. Marker genes for each cell type were calculated using the Wilcoxon Rank Sum test returning only genes that are present in a minimum of 25% of the analyzed cluster 5. The similar goblet and Paneth cells were further differentiated by subsetting and reclustering the cell population containing both cell types in an unbiased manner. This resulted in a small group of cells separated from the rest that were enriched with canonical Paneth markers, Lyz1, Defa17, Defa22, Defa24, and Ang4. These cells were annotated as Paneth cells and the rest as goblet cells (Supplemental Figure 3B). GSEA 4.3.3 (RRID:SCR_003199) was used for pathway enrichment 21. Pathway gene lists were obtained through the Molecular Signatures Database (MSigDB: RRID:SCR_016863). Pathway gene expression heatmaps were plotted using Seurat’s DoHeatmap function using values scaled on the mean.

Data Availability

All data are included in the Supplementary Information or available from the authors, as are unique reagents used in this Article. The raw numbers for charts and graphs are available in the Source Data file whenever possible. Source data are provided with this paper. The scRNA-seq data generated in this study were deposited at the Gene Expression Omnibus (RRID:SCR_005012) under accession number GSE294390 (GSM8903735, GSM8903736, GSM8903737, GSM8903738). The hyperlinks for these data are:
GSE294390[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE294390](study)
GSM8903735[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM8903735](WT)
GSM8903736[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM8903736](CDC42 KO)
GSM8903737[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM8903737](Kras)
GSM8903738[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM8903738](CDC42 KO/Kras)

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Cdc42 is a key regulator of epithelial polarity and stem cell maintenance, while oncogenic Kras drives proliferation and\ndisrupts epithelial homeostasis. Although human data suggest these mutations are mutually exclusive to colon cancer,\nZheng Zhang and colleagues use inducible Villin-CreER and Olfm4-CreER mouse models to show that combined Cdc42\ndeletion and Kras activation cause severe intestinal defects resembling necrotizing enterocolitis (NEC), including villous\ndisruption, inflammation, loss of intestinal stem cells (ISCs), and enterocyte necrosis. The authors highlight the synergistic\neffects of Cdc42 loss and oncogenic Kras activation in the intestinal epithelium. Further single-cell RNA-seq reveals altered\npolarity programs, hyperactive IL-1 signaling, and necroptosis, which are partially reversible by targeting YAP, IL-1R, or\nnecroptosis pathways. Overall, this study highlights a pathogenic mechanism where disrupted polarity cooperates with\noncogenic signaling to impair intestinal homeostasis, providing insight into NEC-like pathology and epithelial vulnerability\nunder dual stress conditions. However, the authors need to address following issues before the manuscript can be\nconsidered for publication.\n1. Is the lethality more closely related to damage in specific regions (e.g., small intestine vs. colon)? Although the authors\nmention that both are affected in Fig. 1, are there any regional differences?\n2. Fig. 2B-C shows that the numbers of goblet cells and enteroendocrine cells remain unchanged. However, given the\ndisrupted epithelial architecture and loss of polarity, could their functions also be impaired? Have the authors considered\nperforming functional analyses of these cells, such as assessing mucus secretion or hormone expression?\n3. In Fig 2F, the authors report disrupted polarity in the villus region; however, a systematic analysis of key polarity proteins\n(e.g., Par3, aPKC, E-cadherin) is lacking. A more comprehensive assessment of polarity markers is recommended to\nstrengthen the evidence supporting the loss of epithelial polarity, a central phenotype in this study.\n4. Additionally, could the mislocalization of Paneth cells be a primary contributor to the observed reduction in intestinal stem\ncells (ISCs)? The authors are encouraged to explore the causal relationship between these two phenomena, potentially by\nassessing Paneth cell function,such as lysozyme secretion, to determine whether their niche-supporting role is\ncompromised.\n5. In Fig S3A, the cluster annotations need clarification, including the marker genes used to distinguish each cell subtype.\nFor example, how were Paneth and goblet cells differentiated at the single-cell level? The macrophage cluster in the UMAP\nplot also appears unusual. Was CD68 or any other specific macrophage marker highly expressed in that cluster?\n6. In Fig 3A, the GSEA analysis was conducted on all intestinal epithelial clusters combined. Are there subtype-specific\ndifferences in pathway enrichment across different intestinal cell types?\n7. While the scRNA-seq data point to activated inflammatory signaling, necroptosis, and disrupted adhesion junction\npathways, it remains unclear whether these changes are cell-autonomous or driven by immune cell-derived\nmicroenvironmental cues. Is there any evidence to distinguish epithelial-intrinsic changes from immune-mediated effects?\n8. Given the strong enrichment of tnf, tnfrsf23, and bok in the enterocyte cluster, have the authors tested whether these\npathways directly cause cell death? For instance, could inhibition of TNF signaling (e.g., anti-TNF antibodies) or genetic\nknockout strategies rescue the enterocyte death phenotype?\n9. The authors argue that the absence of cleaved caspase-3 suggests non-apoptotic cell death, but have they considered\nother forms of programmed cell death, such as pyroptosis? Since caspase-1 can also trigger pyroptosis beyond its role in IL-\n1β maturation, this warrants further exploration.\n10. The authors report a general increase in immune cell populations. Have they examined the spatial distribution of these\nimmune cells within the intestinal tissue? Do specific immune cell types (e.g., T cells vs. macrophages) localize to distinct\npathological regions, which could help better understand the inflammatory response?\n11. Can the authors further explore whether YAP signaling plays a role in regulating inflammation or immune responses in\nthis model? While YAP is well known for its role in stem cell biology, it has also been implicated in immune modulation. An\nintegrated discussion of YAP signaling with inflammation and necroptosis pathways could be valuable.\n12. Although the number of enterocytes and ISCs appears restored upon treatment, it remains unclear whether their\nfunctions are fully recovered. Have the authors considered assessing the functional status of these cells, such as nutrient\nabsorption, stemness gene expression profiles, or clonogenic potential, to provide deeper biological relevance to the\nobserved rescue?", "reviewer_3": "The authors demonstrate that simultaneous deletion of Cdc42 and activation of oncogenic Kras (G12D) in intestinal\nepithelial cells or intestinal stem cells induces NEC-like pathology in mice, challenging traditional views of oncogenic\ncooperation in intestinal transformation. This finding is novel. However, the study leans heavily on phenotypic observations\nrather than mechanistic insights.\nWhile Cdc42 regulates epithelial polarity and Kras mutations drive intestinal tumorigenesis, the dual genetic manipulation in\nepithelial or stem cells to model NEC-like pathology questions its relevance to human disease. Human NEC, primarily\naffecting premature infants, is linked to underdeveloped barrier function, microbial dysbiosis, immature immunity, and\nfeeding practices, not somatic genetic mutations. Although the authors reference TCGA data showing mutual exclusivity\nbetween KRAS mutations and polarity gene defects in colon cancer, it is unclear if similar alterations occur in NEC patients.\nTo improve clinical relevance, the authors should analyze human NEC tissue samples for KRAS mutations or expression\nchanges in polarity regulators like CDC42, DLG5, or Scrib.\nThe single-cell transcriptomic analysis lacks statistical robustness. In Figure 3, GSEA plots omit adjusted P-values, and\nnecroptosis signaling enrichment in the Cdc42 KO/Kras group relies on just three genes with a normalized enrichment score\n(NES) below 1.5. This is insufficient to confirm pathway activation. The authors should reanalyze the data with proper\nstatistical corrections (e.g., FDR-adjusted P-values) and ensure NES exceeds 1.5 to meet standard thresholds for pathway\nenrichment.\nTreatment with necroptosis inhibitor (Nec-1s), IL-1R antagonist (Anakinra), and YAP inhibitor (Verteporfin) reduces NEC-like\nphenotypes in Cdc42 KO/Kras mice, but the relationships between IL-1 signaling, necroptosis, and YAP activation remain\nambiguous. Are these independent responses to polarity loss and oncogenic stress, or part of a sequential cascade?\nAdditional experiments clarifying the upstream–downstream dynamics of these pathways would strengthen the manuscript.\nThe study uses both epithelial cell–specific and intestinal stem cell–specific double mutant models, both exhibiting NEC-like\npathology. However, it fails to clarify whether these defects stem from aberrant intestinal stem cell behavior or differentiated\nepithelial cells. Which model better reflects human NEC? The phenotype-driven nature of the study would benefit from\ndeeper investigation into the cellular origin of these defects to enhance mechanistic understanding."}, "author_response": "Responses to the reviewers’ critiques:\nReviewer #1\nWe thank the reviewer for appreciating that “this study highlights a pathogenic mechanism where\ndisrupted polarity cooperates with oncogenic signaling to impair intestinal homeostasis, providing\ninsight into NEC-like pathology and epithelial vulnerability under dual stress conditions”. The\ninsightful critiques helped us improve the manuscript significantly.\n1. “Is the lethality more closely related to damage in specific regions (e.g., small intestine vs.\ncolon)? Although the authors mention that both are affected in Fig. 1, are there any regional\ndifferences?”\nAnswer: We provide the H&E and IF staining of additional regions of small intestine and colon in\nSupplementary Figure 1, which demonstrates that similar morphological defects to that found in\nduodenum are also present in the distal small intestine (ileum) and the colon of the Cdc42 KO/Kras\nmice. The results also show that while loss of polarity and necroptosis occur in both small intestine\nand colon of the double mutant mice, the most severe cell death/damages occur in the small\nintestine.\n2. “Fig. 2B-C shows that the numbers of goblet cells and enteroendocrine cells remain unchanged.\nHowever, given the disrupted epithelial architecture and loss of polarity, could their functions also\nbe impaired? Have the authors considered performing functional analyses of these cells, such as\nassessing mucus secretion or hormone expression?”\nAnswer: To consider potential effect on goblet cells and enteroendocrine cells, we have compared\nmucin secretion related genes in goblet cells and hormone expression related genes in\nenteroendocrine cells by pathway enrichment analyses of the single-cell RNA-seq data. The\nanalysis did not detect significant overall changes in these pathways between Cdc42 KO/Kras cells\nand WT cells (Supplementary Figure 2B). We would like to carry out mucus and hormone secretion\ntests in future studies.\n3. “In Fig 2F, the authors report disrupted polarity in the villus region; however, a systematic\nanalysis of key polarity proteins (e.g., Par3, aPKC, E-cadherin) is lacking. A more comprehensive\nassessment of polarity markers is recommended to strengthen the evidence supporting the loss of\nepithelial polarity, a central phenotype in this study.”\nAnswer: We now include additional polarity marker analyses: E-cadherin staining is shown in Figure\n2B, Par3, aPKC, and Scribble staining are presented in Supplementary Figure 2C. These results\nfurther confirm that polarity is disrupted in the intestinal epithelium of Cdc42 KO/Kras mice.\n4. “Additionally, could the mislocalization of Paneth cells be a primary contributor to the observed\nreduction in intestinal stem cells (ISCs)? The authors are encouraged to explore the causal\nrelationship between these two phenomena, potentially by assessing Paneth cell function, such as\nlysozyme secretion, to determine whether their niche-supporting role is compromised.”\nAnswer: We agree that Paneth cells provide an important niche support for ISCs. However, multiple\nstudies have demonstrated that Paneth cells are dispensable for ISC survival and function\n(reviewed in Michaela Quintero and Linda C. Samuelson, Cellular and Molecular Gastroenterology\nand Hepatology, 2025). Consistent with this, mislocalization of Paneth cells alone is not sufficient to\ncause ISC depletion in our mouse models, since in our rescue experiments with Nec-1s, IL1R\ninhibitor, or YAP inhibitor, ISC recovery was observed despite of persistent Paneth cell\nmislocalization. We now discuss our findings that Paneth cell mislocalization is possibly not the\nprimary cause of ISC loss in our model, in the context of the above cited review article.\n5. “In Fig S3A, the cluster annotations need clarification, including the marker genes used to\ndefine each cell subtype. For example, how were Paneth and goblet cells differentiated at the\nsingle-cell level? The macrophage cluster in the UMAP plot also appears unusual. Was CD68 or\nany other specific macrophage marker highly expressed in that cluster?”\nAnswer: We have included a complete list of the marker genes used to define each cell type in the\nUMAP (provided as a TXT file in Supplemental Materials). In addition, the major marker genes for\neach cell type are shown in a dot plot (Supplementary Figure 3C). The criteria used to distinguish\nPaneth cells from goblet cells is provided in Supplementary Figure 3B: The similar goblet and\nPaneth cells were further differentiated by subsetting and reclustering the cell population containing\nboth cell types in an unbiased manner. This resulted in a small group of cells separated from the\nrest that were enriched with canonical Paneth markers, Lyz1, Defa17, Defa22, Defa24, and Ang4.\nThese cells were annotated as Paneth cells and the rest as goblet cells. The main UMAP was\nreannotated to reflect these cell identities (Supplementary Figure 3A).\nFor the macrophage cluster, we have observed high expression of macrophage-specific markers,\nincluding CD68, CD14, and IL1B, confirming the cluster identity (Supplementary Figure 3C).\n6. “In Fig 3A, the GSEA analysis was conducted on all intestinal epithelial clusters combined. Are\nthere subtype-specific differences in pathway enrichment across different intestinal cell types?”\nAnswer: We have performed various pathway enrichment analyses including acute inflammation,\nactin filament, response to wound, necroptosis, cellular respiration, and cell–cell junction, in three\nintestinal cell subtypes: enterocytes, ISCs/TA cells, and Paneth cells, as shown in Supplementary\nFigure 3. Most pathway changes are consistent across the different cell subtypes, with a few\nexceptions - for example, cellular respiration in ISCs/TA cells does not appear to change.\n7. “While the scRNA-seq data point to activated inflammatory signaling, necroptosis, and disrupted\nadhesion junction pathways, it remains unclear whether these changes are cell-autonomous or\ndriven by immune cell-derived microenvironmental cues. Is there any evidence to distinguish\nepithelial-intrinsic changes from immune-mediated effects?”\nAnswer: To address this question, we have examined IL1 and TNF expression in IL1R inhibitory\nantibody “rescued” samples, which showed that immune cell-derived microenvironmental cues were\neffectively suppressed by IL1R inhibition in Cdc42 KO/Kras mice (Supplementary Figure 6A). As\nshown in Figure 6, necroptosis was rescued by anti-IL1R treatment that suppresses inflammation.\nIn contrast, the epithelial-intrinsic defects such as disrupted polarity marked by Par3 and\nNa+K+ATPase expression/organization (Supplementary Figure 6A, Figure 6D) and altered\nepithelial morphology (Figure 6A), were not restored. These results suggest that inflammatory\nsignaling and necroptosis are largely driven by inflammation–derived microenvironmental cues,\nwhereas disrupted adhesion junction and polarity pathways are epithelial-intrinsic. We now make\nclear of this point in the text.\n8. “Given the strong enrichment of tnf, tnfrsf23, and bok in the enterocyte cluster, have the authors\ntested whether these pathways directly cause cell death? For instance, could inhibition of TNF\nsignaling (e.g., anti-TNF antibodies) or genetic knockout strategies rescue the enterocyte death\nphenotype?”\nAnswer: To address this question, we treated Cdc42 KO/Kras mice with an anti–TNFα antibody and\nfound that inhibition of TNFα effectively rescued enterocyte necroptosis, cell death, proliferation\ndefects, and ISC abnormalities (Supplementary Figure 6C). These results indicate that the strongly\nenriched TNFα signaling signature in the enterocyte cluster directly contributes to the observed\nenterocyte death phenotype.\n9. “The authors argue that the absence of cleaved caspase-3 suggests non-apoptotic cell death, but\nhave they considered other forms of programmed cell death, such as pyroptosis? Since caspase-1\ncan also trigger pyroptosis beyond its role in IL-1β maturation, this warrants further exploration.”\nAnswer: We have examined cleaved gasdermin D in the mutants and found that it is upregulated in\nCdc42 KO/Kras mice but is not rescued by inhibition of necroptosis (Supplementary Figure 5B),\nindicating an activated pyroptosis pathway. This is consistent with our observation that although\ninhibition of necroptosis alone reduced cell death and extended the survival of Cdc42 KO/Kras mice\n(Figure 5), approximately 60% of the mice treated with Nec-1s still had delayed death (Figure 5I),\nsuggesting that unresolved pyroptosis may also contribute to lethality in Cdc42 KO/Kras mice. We\nnow include a discussion of this possibility.\n10. “The authors report a general increase in immune cell populations. Have they examined the\nspatial distribution of these immune cells within the intestinal tissue? Do specific immune cell types\n(e.g., T cells vs. macrophages) localize to distinct pathological regions, which could help better\nunderstand the inflammatory response?”\nAnswer: We have performed T cell and macrophage marker staining as well as TNFα staining to\nexamine the spatial distribution of cytokine and these immune cells within the intestinal tissue\n(Supplementary Figure 4B). In Cdc42 KO/Kras mice, the increased TNFα signal, T cells, and\nmacrophages were primarily localized in the top region of the villi, including capillaries and lacteals,\nsuggesting that the immune cells are enriched broadly in the epithelial villi.\n11. “Can the authors further explore whether YAP signaling plays a role in regulating inflammation\nor immune responses in this model? While YAP is well known for its role in stem cell biology, it has\nalso been implicated in immune modulation. An integrated discussion of YAP signaling with\ninflammation and necroptosis pathways could be valuable.”\nAnswer: To this end, we have examined the inflammatory response in YAP inhibitor rescued\nsamples and found that YAP inhibition effectively attenuated immune activation in the Cdc42\nKO/Kras small intestine (Supplementary Figure 7A), suggesting that YAP signaling contributes to\nthe regulation of inflammation in this context. On the other hand, IL1R inhibitor or Nec-1s has no\nimpact on the elevated nuclear YAP expression in Cdc42 KO/Kras mice crypts (Supplementary\nFigure 7C, marked by arrow), suggesting that YAP signaling is upstream of the\nnecroptosis/inflammation feed-forward loop. We now discuss this in the context of the working\nmodel (Figure 8).\n12. “Although the number of enterocytes and ISCs appears restored upon treatment, it remains\nunclear whether their functions are fully recovered. Have the authors considered assessing the\nfunctional status of these cells, such as nutrient absorption, stemness gene expression profiles, or\nclonogenic potential, to provide deeper biological relevance to the observed rescue?”\nAnswer: Thanks for the excellent suggestion. While we intend to further expand our studies to the\nfunctional characterization of various cell populations beyond that by scRNAseq in future studies,\nwe have performed an enteroid colony growth assay of the Nec-1s rescued crypt samples. We found\nthat while Nec-1s treatment restored the survival of Cdc42 KO/Kras ISCs and allowed them to form\nenteroid spheres compared to untreated Cdc42 KO/Kras ISCs, their clonogenic potential was not fully\nrescued (Supplementary Figure 5D). This observation suggests that the “rescue” of the crypt cell\nnumber/survival is incomplete, and additional defects to the ISCs caused by the genetic mutations\nare beyond those of cell number/survival function. We now discuss this in the text.\nReviewer #3\nI thank the reviewer for appreciating that our study “demonstrates that simultaneous deletion of\nCdc42 and activation of oncogenic Kras in intestinal epithelial cells or intestinal stem cells induces\nNEC-like pathology in mice, challenging traditional views of oncogenic cooperation in intestinal\ntransformation. This finding is novel.” We have performed additional experimentations and\nclarified key issues about the mechanistic insights and human NEC phenotype comparisons based\non the reviewer’s very helpful suggestions.\n1. “The study leans heavily on phenotypic observations rather than mechanistic insights.”\nAnswer: Our study of two combined oncogenic cues, i.e. loss of polarity by Cdc42 KO and\noncogenic KRas expression, in mouse models led to an unexpected observation that mimics many\nof the human NEC-like phenotypes. With additional experimentations performed to address both\nreviewers’ suggestions, the study has established a mechanistic association of the polarity\nloss/oncogenic Kras cues with almost all the major effects found in human NEC. We demonstrate\nby phenotypic “rescues” using inhibitors of IL1R, TNF, necroptosis, or YAP signaling that the\ncombined oncogenic cues elicit a inflammation response leading to necroptosis of the intestinal\nenterocytes, which further feed forward to the inflammatory signals to exacerbate the NEC-like\nphenotypes. Mechanistically, The IL1, TNF, necroptosis, as well as YAP signaling, are causal to\nthis feed-forward loop. We now clearly depict a mechanistic summary of this model in Figure 8\n“Graphic Model”. We believe that these pathway-specific causal characterizations provide new\nmechanistic insights into the observed phenotypes.\n2. ”While Cdc42 regulates epithelial polarity and Kras mutations drive intestinal tumorigenesis, the\ndual genetic manipulation in epithelial or stem cells to model NEC-like pathology questions its\nrelevance to human disease. Human NEC, primarily affecting premature infants, is linked to\nunderdeveloped barrier function, microbial dysbiosis, immature immunity, and feeding practices, not\nsomatic genetic mutations. Although the authors reference TCGA data showing mutual exclusivity\nbetween KRAS mutations and polarity gene defects in colon cancer, it is unclear if similar\nalterations occur in NEC patients. To improve clinical relevance, the authors should analyze human\nNEC tissue samples for KRAS mutations or expression changes in polarity regulators like CDC42,\nDLG5, or Scrib.”\nAnswer: We apologize for this confusion. We agree that somatic genetic mutations are not the\nunderlying cause for human NEC. Our work does not implicate somatic genetic mutations as a\ncause for NEC, nor is it focused on studying human NEC. Rather, we report an unexpected finding\nthat the combined oncogenic cues, i.e. loss of polarity and Kras mutations, lead to NEC-like\nphenotypes, suggesting a mutual exclusivity between KRAS mutations and polarity gene defects\nfound in colon cancer.\nTo improve the relevance of our work to human NEC, we have analyzed human intestine tissues\nfrom normal and NEC patients by examining the polarity markers (Na⁺/K⁺-ATPase and Scribb),\nthe enterocyte marker ALPi, and the adhesion marker E-cadherin. We observed similar polarity and\nadhesion defects in the NEC tissues as seen in our Cdc42 KO/Kras mouse model. Enterocytes,\nthe predominant cell type in the NEC small intestine, were also severely impaired, closely\nrecapitulating the epithelial defects observed in the mouse model, supporting that our mouse model\nis “NEC-like”.\nWe now add a new figure describing all the human NEC phenotypes and changes in\nSupplementary Figure 9. In addition, we have revised the text in the Discussion and the Limitation\nof the Work that the current studies do not mean to implicate somatic mutations, such as a loss of\npolarity gene and oncogenic Kras, in human NEC; rather, the study is about the combined genetic\nmutations causing NEC-like effects.\n3. “The single-cell transcriptomic analysis lacks statistical robustness. In Figure 3, GSEA plots omit\nadjusted P-values, and necroptosis signaling enrichment in the Cdc42 KO/Kras group relies on just\nthree genes with a normalized enrichment score (NES) below 1.5. This is insufficient to confirm\npathway activation. The authors should reanalyze the data with proper statistical corrections (e.g.,\nFDR-adjusted P-values) and ensure NES exceeds 1.5 to meet standard thresholds for pathway\nenrichment.”\nAnswer: We have expanded the necroptosis gene set for the pathway enrichment analysis, which\nresulted in an updated NES of 1.51 with an adjusted P value of 0.03. Additionally, we have replaced\nthe P values in Figure 3 with FDR-adjusted P values to improve statistical robustness.\n4. “Treatment with necroptosis inhibitor (Nec-1s), IL-1R antagonist (Anakinra), and YAP inhibitor\n(Verteporfin) reduces NEC-like phenotypes in Cdc42 KO/Kras mice, but the relationships between\nIL-1 signaling, necroptosis, and YAP activation remain ambiguous. Are these independent\nresponses to polarity loss and oncogenic stress, or part of a sequential cascade? Additional\nexperiments clarifying the upstream–downstream dynamics of these pathways would strengthen\nthe manuscript.”\nAnswer: This question is related to reviewer #1, question #11. We have examined the inflammatory\nresponse in YAP inhibitor rescued samples and found that YAP inhibition attenuates immune\nactivation in the Cdc42 KO/Kras intestine (Supplementary Figure 7A). An examination of nuclear\nYAP expression (Supplementary Figure 7C, marked by arrow) in Nec-1s rescued samples and in\nIL1R antibody rescued samples shows loss of Cdc42/polarity-elicited YAP activation is causal for\nthe subsequent hyperinflammation and necroptosis effects, and suppression of IL1-mediated\ninflammation and necroptosis are circularly associated such that inflammation is upstream of\nnecroptosis, which can feed forward to further exacerbate inflammation. We now discuss this in the\ncontext of the working model (Figure 8).\n5. “The study uses both epithelial cell–specific and intestinal stem cell–specific double mutant\nmodels, both exhibiting NEC-like pathology. However, it fails to clarify whether these defects stem\nfrom aberrant intestinal stem cell behavior or differentiated epithelial cells. Which model better\nreflects human NEC? The phenotype-driven nature of the study would benefit from deeper\ninvestigation into the cellular origin of these defects to enhance mechanistic understanding.”\nAnswer: Our studies by using the intestinal stem cell Olmf4-CreER driver to delete Cdc42 gene and\nactivate oncogenic Kras showed similar defects in inflammation, loss of polarity/junctional defects,\nenterocyte necroptosis, etc, as by using the pan-intestinal epithelial Villin-CreER driver. To better\ncompare with human NEC phenotypes, we further analyzed human NEC samples and found that the\ncrypt regions are relatively intact, showing normal proliferation and TA cell populations. The main\ndefects were observed in the villus regions, characterized by increased immune activation,\nnecroptosis, and cell death (Supplementary Figure 9). Therefore, both models of the Cdc42 KO/Kras\nmice reflect similar epithelial cell and inflammatory defects seen in human NEC, suggesting that while\nthe intestinal epithelia necroptosis and inflammation are the manifested phenotypes, original defects\nin ISCs can be propagated through differentiation to mimic that of the NEC-like effect in the epithelium.\nWe now discuss this point in Discussion."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed most of the technical issues. Through supplementary experiments and in-depth analysis, they\nhave substantially strengthened the study's mechanistic insights. The reviewer has not additional comments.", "reviewer_3": "No further comment."}, "author_response": ""}]

Decision letter