CRISPR activation screens identify the SWI/SNF ATPases as suppressors of ferroptosis.

Ferroptosis is an iron-dependent cell death mechanism characterized by the accumulation of toxic lipid peroxides and cell membrane rupture. GPX4 (glutathione peroxidase 4) prevents ferroptosis by reducing these lipid peroxides into lipid alcohols. Ferroptosis induction by GPX4 inhibition has emerged as a vulnerability of cancer cells, highlighting the need to identify ferroptosis regulators that may be exploited therapeutically. Through genome-wide CRISPR activation screens, we identify the SWI/SNF (switch/sucrose non-fermentable) ATPases BRM (SMARCA2) and BRG1 (SMARCA4) as ferroptosis suppressors. Mechanistically, they bind to and increase chromatin accessibility at NRF2 target loci, thus boosting NRF2 transcriptional output to counter lipid peroxidation and confer resistance to GPX4 inhibition. We further demonstrate that the BRM/BRG1 ferroptosis connection can be leveraged to enhance the paralog dependency of BRG1 mutant cancer cells on BRM. Our data reveal ferroptosis induction as a potential avenue for broadening the efficacy of BRM degraders/inhibitors and define a specific genetic context for exploiting GPX4 dependency.

Breast Cancer Knowledge Among Amish and Mennonite Women.

Breast cancer is the most common cancer diagnosis for women in the USA and ranks second in cancer-related deaths. Disproportionately higher breast cancer rates can be found in rural and Appalachian regions due to several social drivers of health, including poverty, access to healthcare, and lack of culturally sensitive health education. Amish and Mennonite communities, religious groups with distinct cultural practices and beliefs, experience lower mammography screening and higher breast cancer mortality rates (among Amish women). This study focuses on knowledge about breast cancer and causes of cancer among Amish and Mennonite women. A total of 473 women participated in the study at 26 separate women's health clinics throughout Ohio, consisting of 348 Amish and 121 Mennonite women, the largest study conducted on breast cancer knowledge spanning dozens of communities. Statistically significant differences were found in total knowledge scores between Amish and Mennonite women (rpb = .178, n = 466, p = .007), with Amish women having lower scores and stronger beliefs in myths associated with breast cancer cause and symptoms (χ(1) = 7.558, p = .006). Both groups often provided scientifically accurate descriptions of cancer etiology. The majority of participants underestimated breast cancer risk, highlighting the need for culturally appropriate health education programs that consider numeracy and health literacy. By implementing targeted interventions and fostering partnerships with community stakeholders using a multifaceted approach that incorporates cultural sensitivity, community engagement, and collaboration, significant progress can be made towards reducing breast cancer disparities and improving health outcomes.

Early clinical markers of overweight/obesity onset and resolution by adolescence.

OBJECTIVES: We examined how combinations of clinical indicators at various ages predict overweight/obesity development, as well as resolution, by 10-11 and 14-15 years of age. METHODS: Data were derived from Birth (N = 3469) and Kinder (N = 3276) cohorts of the Longitudinal Study of Australian Children, followed from ages 2-3 and 4-5 years, respectively. Every two years, 25 potential obesity-relevant clinical indicators were quantified. Overweight/obesity was defined using International Obesity Taskforce cutpoints at 10-11 years and 14-15 years. RESULTS: In both cohorts, three factors predicted both development and resolution of overweight/obesity in multivariable models. Among normal weight children, increased odds of developing overweight/obesity were associated with higher child (odd ratio (OR) 1.67-3.35 across different study waves) and maternal (OR 1.05-1.09) BMI, and inversely with higher maternal education (OR 0.60-0.62, when assessed at age 2-7 years). Lower odds of resolving existing overweight/obesity were related with higher child (OR 0.51-0.79) and maternal (OR 0.89-0.95) BMI, and inversely with higher maternal education (OR 1.62-1.92, when assessed at age 2-5 years). The prevalence of overweight/obesity at the age of 14-15 years was 13% among children with none of these risk factors at age 6-7 years, compared with 71% among those with all 3 risk factors (P < 0.001). CONCLUSIONS: From early childhood onwards, child and maternal BMI and maternal education predict overweight/obesity onset and resolution by adolescence. A simple risk score, easily available to child health clinicians, could help target treatment or prevention.

ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Growth in KEAP1-Mutant Lung Cancer.

Mutations in KEAP1 and NFE2L2 (encoding the protein Nrf2) are prevalent in both adeno and squamous subtypes of non-small cell lung cancer, as well as additional tumor indications. The consequence of these mutations is stabilized Nrf2 and chronic induction of a battery of Nrf2 target genes. We show that knockdown of Nrf2 caused modest growth inhibition of cells growing in two-dimension, which was more pronounced in cell lines expressing mutant KEAP1. In contrast, Nrf2 knockdown caused almost complete regression of established KEAP1-mutant tumors in mice, with little effect on wild-type (WT) KEAP1 tumors. The strong dependency on Nrf2 could be recapitulated in certain anchorage-independent growth environments and was not prevented by excess extracellular glutathione. A CRISPR screen was used to investigate the mechanism(s) underlying this dependence. We identified alternative pathways critical for Nrf2-dependent growth in KEAP1-mutant cell lines, including the redox proteins thioredoxin and peroxiredoxin, as well as the growth factor receptors IGF1R and ERBB3. IGF1R inhibition was effective in KEAP1-mutant cells compared with WT, especially under conditions of anchorage-independent growth. These results point to addiction of KEAP1-mutant tumor cells to Nrf2 and suggest that inhibition of Nrf2 or discrete druggable Nrf2 target genes such as IGF1R could be an effective therapeutic strategy for disabling these tumors. SIGNIFICANCE: This study identifies pathways activated by Nrf2 that are important for the proliferation and tumorigenicity of KEAP1-mutant non-small cell lung cancer.

An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells.

The KDM5 family of histone demethylases catalyzes the demethylation of histone H3 on lysine 4 (H3K4) and is required for the survival of drug-tolerant persister cancer cells (DTPs). Here we report the discovery and characterization of the specific KDM5 inhibitor CPI-455. The crystal structure of KDM5A revealed the mechanism of inhibition of CPI-455 as well as the topological arrangements of protein domains that influence substrate binding. CPI-455 mediated KDM5 inhibition, elevated global levels of H3K4 trimethylation (H3K4me3) and decreased the number of DTPs in multiple cancer cell line models treated with standard chemotherapy or targeted agents. These findings show that pretreatment of cancer cells with a KDM5-specific inhibitor results in the ablation of a subpopulation of cancer cells that can serve as the founders for therapeutic relapse.

Tankyrase Inhibition Causes Reversible Intestinal Toxicity in Mice with a Therapeutic Index < 1.

Activated Wnt/ß-catenin signaling is frequently associated with colorectal cancer. Wnt inhibitors, including tankyrase inhibitors, are being explored as potential anticancer agents. Wnt signaling is also critical for intestinal tissue homeostasis, and Wnt inhibitors have been shown to cause intestinal toxicity in mice by affecting intestinal stem cells. This study sought to characterize the intestinal toxicity of tankyrase inhibitors, including reversibility, and to assess their therapeutic index. Novel tankyrase inhibitor G-631 caused dose-dependent intestinal toxicity with a therapeutic index < 1 after 14 days of dosing in mice. At a tolerated subtherapeutic dose level, the intestinal toxicity was composed of enteritis characterized by villus blunting, epithelial degeneration, and inflammation, which fully reversed after 14 days of recovery. Doubled exposure showed weak antitumor activity in a xenograft colorectal cancer model but also caused more severe intestinal toxicity characterized by multifocal-regionally extensive necrotizing and ulcerative enteritis leading to morbidity or moribundity in some animals. This toxicity was only partially reversed after 14 days of recovery, with evidence of crypt and villus regeneration, mildly blunted villi, and/or scarring in association with chronic inflammation of the submucosa. Therefore, the clinical utility of tankyrase inhibitors is likely limited by the on-target intestinal toxicity and a therapeutic index < 1 in mice.

A novel tankyrase small-molecule inhibitor suppresses APC mutation-driven colorectal tumor growth.

Most colorectal cancers (CRC) are initiated by mutations of APC, leading to increased ß-catenin-mediated signaling. However, continued requirement of Wnt/ß-catenin signaling for tumor progression in the context of acquired KRAS and other mutations is less well-established. To attenuate Wnt/ß-catenin signaling in tumors, we have developed potent and specific small-molecule tankyrase inhibitors, G007-LK and G244-LM, that reduce Wnt/ß-catenin signaling by preventing poly(ADP-ribosyl)ation-dependent AXIN degradation, thereby promoting ß-catenin destabilization. We show that novel tankyrase inhibitors completely block ligand-driven Wnt/ß-catenin signaling in cell culture and display approximately 50% inhibition of APC mutation-driven signaling in most CRC cell lines. It was previously unknown whether the level of AXIN protein stabilization by tankyrase inhibition is sufficient to impact tumor growth in the absence of normal APC activity. Compound G007-LK displays favorable pharmacokinetic properties and inhibits in vivo tumor growth in a subset of APC-mutant CRC xenograft models. In the xenograft model most sensitive to tankyrase inhibitor, COLO-320DM, G007-LK inhibits cell-cycle progression, reduces colony formation, and induces differentiation, suggesting that ß-catenin-dependent maintenance of an undifferentiated state may be blocked by tankyrase inhibition. The full potential of the antitumor activity of G007-LK may be limited by intestinal toxicity associated with inhibition of Wnt/ß-catenin signaling and cell proliferation in intestinal crypts. These results establish proof-of-concept antitumor efficacy for tankyrase inhibitors in APC-mutant CRC models and uncover potential diagnostic and safety concerns to be overcome as tankyrase inhibitors are advanced into the clinic.

Ubiquitin ligase RNF146 regulates tankyrase and Axin to promote Wnt signaling.

Canonical Wnt signaling is controlled intracellularly by the level of ß-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates ß-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.

Inhibition of Wnt signaling by Dishevelled PDZ peptides.

Dishevelled proteins are key regulators of Wnt signaling pathways that have been implicated in the progression of human cancers. We found that the binding cleft of the Dishevelled PDZ domain is more flexible than those of canonical PDZ domains and enables recognition of both C-terminal and internal peptides. These peptide ligands inhibit Wnt/beta-catenin signaling in cells, showing that Dishevelled PDZ domains are potential targets for small-molecule cancer therapeutics.