Jul 2026· Journal of Immunology· Vol 215· 0 citations
TL;DR
The in vitro proliferation of AKP-M4 cells, but not KRAS wild-type SNU-1079 and SSP-25 cells, was reduced by the KRAS G12D mutation inhibitor MRTX-1133, which supports the development of KRAS mutation inhibitor for CCA treatment.
Abstract
Cholangiocarcinoma (CCA), is an aggressive malignancy often diagnosed at an advanced, inoperable stage, with 5-year survival rate of 3%. KRAS mutations are frequent driver alterations linked to poorer prognosis in this cancer. The KRAS G12C mutation inhibitor Adagrasib has been approved by the FDA for the treatment of advanced non-small-cell lung cancer, indicating that the KRAS mutation is a druggable target. However, KRAS mutant inhibitors have yet to be employed in CCA treatment.
A mouse CCA model, designated AKP, was generated by crossing genetically engineered mouse strains harboring Alb-Cre, LSL-KrasG12D, and p53L/L. Subsequently, the AKP-M4 cell line was derived by serially passaging tumor cells from AKP mice across three generations. Sanger sequencing and immunohistochemical (IHC) staining were used to determine the KRAS G12D mutation and cytokeratin 19, respectively. MTT assay was used to assess the cell proliferation inhibition by MRTX-1133. Immune cell profiles in AKP-M4-bearing mice treated with MRTX-1133 were determined by flow cytometry.
Sanger sequencing confirmed G12D mutation in the KRAS gene in AKP-M4 cells. IHC analysis demonstrated that AKP-M4 tumors expressed cytokeratin 19, a key marker for CCA. The in vitro proliferation of AKP-M4 cells, but not KRAS wild-type SNU-1079 and SSP-25 cells, was reduced by the KRAS G12D mutation inhibitor MRTX-1133. We further assessed the anti-cancer efficacy of MRTX-1133 in AKP-M4-bearing C57BL/6 mice, observing a significant dose-dependent reduction in tumor volume and weight. Flow cytometry analysis showed that MRTX-1133 elevated CD4+ T cells, CD8+ T cells, and M1 macrophages, while decreasing MDSCs and M2 macrophages. Notably, PD-L1 expression on tumor cells was diminished.
KRAS G12D mutant inhibition reduces cancer cell proliferation and stimulates immune cells. This supports the development of KRAS mutation inhibitor for CCA treatment.
National Science and Technology Council, Taiwan
Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Treatment of NSCLC KRAS G12C mutant tumors with the allele-selective sotorasib and adagrasib inhibitors is invariably associated with acquired resistance. The MUC1-encoded oncogenic M1C protein is necessary for self-renewal of NSCLC KRAS mutant cells. We report that treatment of NSCLC KRAS G12C cells with sotorasib induces M1C expression by a STAT1-dependent pathway. In turn, M1C drives the sotorasib resistant phenotype by NF-κB-mediated induction of the epithelial-mesenchymal transition (EMT) and a mucinous gene program. Targeting M1C→NF-κB signaling (i) suppresses EMT and mucin genes, and (ii) reverses sotorasib resistance. Of translational relevance, treatment with a M1C antibody-drug conjugate (ADC) is effective against two sotorasib-resistant NSCLC KRAS G12C cell lines and two patient-derived tumor xenograft models. Analysis of patients with NSCLC KRAS G12C tumors treated with sotorasib/adagrasib and overexpressing MUC1 associates with decreases in overall survival. These findings identify M1C as a key effector of sotorasib resistance and as a target for treatment of patients with refractory NSCLC KRAS G12C mutant tumors.
Mediator complex kinase CDK8 is identified as a driver of resistance towards KRASG12D inhibition in pancreatic ductal adenocarcinoma (PDAC), promoting stromal remodeling and immunosuppression and CDK8 inhibition in resistant tumors re-primes PDAC to anti-CTLA-4 immunotherapy efficacy.
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The syngeneic murine Ff-iCCA model is introduced, providing a platform for functional investigation of cancer cell-TME crosstalk in this molecular subtype, and overcoming the key limitation of prior models relying on human transgenes in immunodeficient hosts.
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EGFR is a key oncogenic driver of lung adenocarcinoma, and resistance to its tyrosine kinase inhibitors has become a major clinical challenge in the treatment of EGFR-mutated LUAD patients. Through preliminary experiments including Western blot and immunohistochemistry staining, RBM15 was identified as an oncogene promoting drug resistance in EGFR-mutant LUAD cell lines PC-9 and HCC827. Analysis of the TCGA-LUAD cohort revealed a positive correlation between RBM15 and EGFR expression, whereas RBM15 gain-of-function was negatively associated with LUAD patient survival. RBM15 expression was higher in EGFR-mutant LUAD cell lines PC-9 and HCC827 compared to non-EGFR-mutant cell line A549. Furthermore, RBM15 was downregulated in PC-9 and HCC827 cells following gefitinib treatment. Knockdown of RBM15 reduced proliferation and promoted apoptosis in PC-9 and HCC827 cells in vitro, while also inhibiting the growth of PC-9 xenograft tumors in mice. Notably, RBM15 overexpression rescued these effects and promoted gefitinib resistance in PC-9 and HCC827 cells. Transcriptomic and metabolomic sequencing analyses of RBM15-knockdown PC-9 cells revealed enrichment in multiple cancer signaling pathways, including mitochondrial fatty acid metabolism. Diacylglycerol kinase ε (DGKE) was identified as a novel interacting protein of RBM15, and RBM15 was found to influence fatty acid metabolism by modulating mitochondrial function. In summary, RBM15 promotes tumorigenic proliferation, suppresses apoptosis, and enhances gefitinib resistance in EGFR-mutant LUAD cells by regulating the EGFR signaling pathway and interacting with DGKE. Based on the interplay among RBM15, EGFR, and downstream DGKE, RBM15 may serve as a promising new therapeutic target for EGFR-mutant LUAD.
Mingsheng Ma, Wei Wang, Xiaoyan Wang et al.· Scientific Reports· 0 citations
Abstract Loss of the 9p21 chromosomal locus, which contains CDKN2A, has been associated with non-response to immune checkpoint inhibition (ICI) across multiple human tumor histologies, yet the underlying mechanisms remain elusive. Given the high frequency of CDK pathway alterations in human brain metastases (BM), especially those arising from triple negative breast cancer (TNBC), we sought to explore the relationship between loss of p16, a CDKN2A isoform, and ICI resistance associated with 9p21 loss. We used CRISPR-Cas9 to generate an isogenic p16-knockout derivative (EMT6 sgRNA3) from ICI-sensitive murine TNBC cell line EMT6, as well as a non-targeting control (EMT6 sgNT). With these cell lines, we established an in vivo model in BALB/c mice to assess intracranial response to anti-PD-1 treatment. Preliminary results revealed a trend toward diminished intracranial ICI efficacy in mice bearing EMT6 sgRNA3 tumors compared to EMT6 sgNT controls (n = 10/group, p=0.08). To validate this finding, we generated ex vivo organotypic spheroids (OTS) from intracranial tumors at survival endpoint and assessed anti-PD-1-induced cell death by lactate dehydrogenase release. Compared to IgG control (10 μg/mL), anti-PD-1 (10 μg/mL) treatment produced a significant increase in cytotoxicity in EMT6 sgNT OTS but not EMT6 sgRNA3 OTS (p=0.001 vs. p=0.76, respectively) after 72 hours. To interrogate the mechanistic basis of these findings, we performed bulk RNAseq on intracranial tumors (n = 4/group), which revealed a significant decrease in expression of chemokines (Cxcl1, Cxcl10, Cxcl11) and immune-related genes (Cd274, Vcam1, Il2ra) in EMT6 sgRNA3 tumors, suggesting that p16 loss is associated with an immunosuppressive microenvironment characterized by decreased innate immune signaling and immune cell recruitment. These findings suggest a role for p16 loss as a candidate functional driver of ICI resistance associated with 9p21 loss in TNBC-BM and warrant further investigation given the need to nominate biomarkers for ICI response to inform patient care.
Varun Sasisekharan, Naema Nayyar, C. Torrini et al.· Neuro-Oncology Advances· 0 citations
Gastric adenocarcinoma (GAC) remains a leading cause of cancer-related mortality, particularly in patients with peritoneal carcinomatosis, for whom effective therapies are limited. We investigated the therapeutic efficacy and molecular mechanism of CYD-4-61, a BAX activator, using human GAC cell lines, patient-derived xenograft models, genetically engineered mouse models, and a syngeneic mouse model. CYD-4-61 potently inhibited tumor cell proliferation, induced apoptosis, and suppressed cancer stem cell-like properties, with enhanced activity in radiation-resistant GAC cells. Mechanistically, CYD-4-61 activated the BAX-caspase pathway, leading to SOX9 protein reduction. Integrated bulk and single-cell transcriptomic analyses identified SOX9-dependent transcriptional programs as major targets of CYD-4-61. Functional rescue experiments together with chromatin immunoprecipitation and CUT&RUN analyses supported CDK4 as a SOX9-regulated gene and demonstrated suppression of the SOX9-CDK4 regulatory axis following CYD-4-61 treatment. In multiple preclinical models, CYD-4-61 significantly inhibited tumor growth and improved the therapeutic response to anti-programmed cell death protein 1 (PD-1) therapy while modulating the tumor immune microenvironment. Clinically, co-expression of SOX9 and CDK4 was associated with diffuse-type GAC and poor patient outcomes. These findings identify the BAX-SOX9-CDK4 axis as an important mechanism contributing to the antitumor activity of CYD-4-61 and provide a strong preclinical rationale for its further development as a therapeutic strategy for aggressive GAC.
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