BACKGROUND & AIMS:As the most abundant memory T cells and major source of tumor necrosis factor α in the intestinal mucosa of Crohn's disease (CD) patients, CD4+ tissue-resident memory T (TRM) cells play a critical role in CD pathogenesis. We investigated the role of metabolic reprogramming in the regulation of proinflammatory and apoptosis-resistant phenotype for CD4+ TRM cells.METHODS:CD4+ TRM cells were collected from intestinal resection tissues from control and CD patients. Transcriptomic and metabolomic analysis were performed to identify metabolic characteristics of CD4+ TRM cells. Enzyme-linked immunosorbent assay and quantitative polymerase chain reaction experiments were used to assess cytokines level in CD4+ TRM cells; activation-induced cell apoptosis rate was evaluated by flow cytometry. Transwell assay and wound healing assay were performed to detect the effect of CD4+ TRM cells on the migration of normal intestinal epithelial cells.RESULTS:Transcriptomic data combined with unbiased metabolomic analysis revealed an increased fatty acid oxidation (FAO) phenotype existed in CD4+ TRM cells from CD patients. The lipidomic data and stable isotope tracer experiments demonstrated that CD4+ TRM cells up-regulated their lipid lipolysis and fatty acid uptake to fuel FAO in CD patients. Mechanistically, the activated nuclear factor kappa B signaling increased transcription of genes involved in lipid lipolysis, fatty acid uptake, and oxidation in CD4+ TRM cells from CD patients. Targeting FAO of CD4+ TRM cells reversed their apoptosis-resistant and proinflammatory phenotype in CD patients.CONCLUSIONS:CD4+ TRM cells process an accelerated FAO mediated by activated nuclear factor kappa B signaling in CD patients; targeting FAO could reverse their apoptosis-resistant and proinflammatory phenotype. These findings shed a new light on the pathogenic mechanism investigation and novel therapy development in CD patients.

Abstract Background and Aim: Ferroptosis is an iron-dependent form of programmed cell death characterized by lipid peroxidation, with the potential as a novel cancer therapy. The application of ferroptosis-inducers has been limited to preclinical studies due to toxic side effects and drug-resistance. Our study aims to explore the metabolic mechanism in regulating ferroptosis-resistance, thereby identifying combinational therapeutic target for ferroptosis-inducers. Methods: The ferroptosis-resistant CRC cell lines were induced by chronic exposure to ferroptosis inducer Erastin and verified by cell viability assay. Metabolomics and transcriptomics were performed to identify the metabolic characteristics in ferroptosis-resistant CRC cells. Quantitative RT-PCR and western blot was applied to confirm the differentially expressed serine-glycine-one-carbon metabolism (SGOC) enzymes. The MTT cell viability assay, colony formation assay, 3D spheroid formation assay and cell derived xenograft (CDX) model were carried out to investigate the therapeutic effect of targeting SGOC key enzyme-phosphoglycerate dehydrogenase (PHGDH) in combination of ferroptosis inducers. Gene set enrichment analysis (GSEA) was performed to identify molecular mechanism for the upregulation of SGOC enzymes based on RNA-seq data from TCGA-COAD cohort. Results: Metabolomic and transcriptomic data revealed that SGOC metabolism was significantly increased in ferroptosis-resistant CRC cells. Moreover, the mRNA and protein level of SGOC key enzyme-PHGDH were remarkably upregulated in ferroptosis-resistant CRC cells. PHGDH inhibitor NCT-503 or knockdown of PHGDH exhibited a great synergistic effect with ferroptosis inducers for CRC cells in vitro or in vivo. Additionally, targeting PHGDH could dramatically restore the sensitivity of ferroptosis-inducers in ferroptosis-resistant cells. The RNA methyltransferase-like 3 (METTL3) was identified as the upstream regulator responsible for the upregulation of PHGDH in an m6A-dependent manner. The ferroptosis stress induced deacetylation of METTL3, thereby promoting its nuclear translocation and enzyme activity. Conclusions: In summary, we have uncovered that CRC cells resisted ferroptosis inducers via METTL3-PHGDH axis. Targeting PHGDH combined with ferroptosis inducers exhibited with synergistic effect for CRC in vitro and in vivo. Our findings identified the metabolic vulnerability in ferroptosis-resistant CRC cells, which could serve as a novel therapeutic target. Citation Format: Guanzhan Liang, Jing Chen, Xiaofeng Wen, Zongjin Zhang, Zexian Chen, Yongle Chen, Zhenyu Xian, Xiaowen He, Xianrui Wu, Ping Lan, Tuo Hu. PHGDH-mediated serine-glycine-one-carbon metabolism drives ferroptosis- and chemotherapy-resistance in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3275.

9095 Background: Combination of tyrosine kinase inhibitor (TKI) and chemotherapy has shown improved clinical outcomes in advanced epidermal growth factor receptor ( EGFR) mutated non-small cell lung cancer (NSCLC) patients. We conducted this phase 3, randomized, controlled trial to further investigate the clinical efficacy and safety of gefitinib combined with chemotherapy in EGFR mutated NSCLC patients with brain metastases. Methods: Treatment-naïve, confirmed brain metastases and EGFR sensitive mutated NSCLC patients were screened from six centers in China. The eligible patients were randomly assigned (1:1) to receive gefitinib alone or gefitinib plus pemetrexed-platinum chemotherapy until intracranial progressive diseases, unacceptable adverse, or any cause of death. Theprimary endpoint was intracranial progression-free survival (iPFS), secondary endpoints were PFS, overall survival, intracranial objective response rate, overall objective response rate, and safety. This study is registered at ClinicalTrials. gov, number NCT01951469. Results: From January 2017 to June 2021, 161 patients were randomly assigned to receive gefitinib (n = 81) or gefitinib plus pemetrexed-platinum chemotherapy (n = 80), the median follow-up time was 18.2 (IQR 11.8-29.7) months. The median intracranial PFS was 15.6 (14.3-16.9) months in gefitinib plus chemotherapy group versus 9.1 (8.0-10.2) months in gefitinib group (HR = 0.36, 95% CI, 0.25-0.53, P < 0.001). Similarly, the median PFS was also significantly longer in gefitinib plus chemotherapy than gefitinib alone (16.3 months vs 9.5 months, P < 0.001). In addition, gefitinib plus chemotherapy had better intracranial objective response rate (85.0% versus 63.0%, P = 0.002) and overall objective response rate (80.0% versus 64.2%, P = 0.035) than gefitinib alone. At the data cutoff, 50.3% of patients (35 patients in gefitinib plus chemotherapy group and 46 patients in gefitinib group) had died. The 3-year OS rate was significantly higher in gefitinib plus chemotherapy group (47.4%, 95% CI 36.3-58.7) than in gefitinib group (24.9%, 95% CI 15.1-34.3, P = 0.003). And median overall survival was 35.0 months (95% CI, 28.8-41.3 months) in gefitinib plus chemotherapy group versus 28.9 months (95% CI, 23.4-34.4 months) in gefitinib group (HR = 0.66, 95% CI, 0.42-1.03, P = 0.065). Grade 3 or worse adverse events were more common in gefitinib plus chemotherapy group (40.0% versus 21.0%, P = 0.010), but most of them were manageable. Conclusions: Inuntreated EGFR mutated NSCLC patients with brain metastases, gefitinib plus chemotherapy significantly improved intracranial PFS, PFS and a tendency of OS than gefitinib alone, and could be the optional first-line treatment. Clinical trial information: NCT01951469.

ABSTRACT Osteoblasts provide a microenvironmental niche for B-cell commitment and maturation in the bone marrow (BM). Any abnormity of osteoblasts function may result in the defect of B lymphopoiesis. Signaling from mechanistic target of rapamycin complex 1 (mTORC1) has been implicated in regulating the expansion and differentiation of osteoblasts. Thus, we raise a hypothesis that mTORC1 signaling in osteoblasts plays a vital role in B-cell development. Inactivation of mTORC1 in osterix-expressing cells (mainly osteoblast lineage) through Osx-Cre-directed deletion of Raptor (an mTORC1-specific component) resulted in a reduction in the total B-cell population in the BM, which was due to a block in early B-cell development from the pro-B to pre-B cell stage. Further mechanistic studies revealed that this defect was the result of reduction of interleukin-7 (IL-7) expression in osterix-expressing immature osteoblasts, which caused the abnormality of IL-7/Stat5 signaling in early B lymphocytes, leading to an increased apoptosis of pre-B plus immature B cells. In vitro and in vivo studies demonstrated that the addition of exogenous IL-7 partially restored B lymphopoiesis in the BM of Raptor mutant mice. Furthermore, total BM cells cultured in conditioned media from Raptor null immature osteoblasts or media with anti-IL-7 neutralizing antibody failed to differentiate into pre-B and immature B cells, indicating that inactivation of mTORC1 in immature osteoblast cannot fully support normal B-cell development. Taken together, these findings demonstrate a novel role for mTORC1 in the regulation of bone marrow environments that support B-cell differentiation via regulating IL-7 expression. © 2017 American Society for Bone and Mineral Research.