Abstract 1671: Targeting histone H3K27 demethylase for anti-tumor activity: Monotherapy and combination with ceralasertib in non-small cell lung carcinoma

作者: Yang, Chih-Chieh ; Chang, Yu-Hsien ; Lin, Kun-Yuan ; Cheng, Shan-Yun ; Chang, Chao-Di ; Hung, Ya-Wen ; Shen, Chien-Chang ; Yu-Ling Lee, Pony ; Wang, Shu-Ping

AbstractLung cancer is the leading cause of cancer-related deaths worldwide, with non-small cell lung carcinoma (NSCLC) accounting for 85% of cases and associated with poor prognosis. The lysine-specific demethylase KDM6A, responsible for removing H3K27 trimethylation at gene promoters and enhancers, is frequently mutated in various cancers. While KDM6A is predominantly considered as a tumor suppressor gene, recent studies suggusted it may support oncogenic transcriptional regulation. To investigate the role of KDM6A in tumorigenesis, we utilized CRISPR-Cas9/sgRNA to knock out (KO) KDM6A in NCI-H1975 NSCLC cells. Phenotypic assessments demonstrated cell cycle arrest with inhibitory effects in cell proliferation with KDM6A-depleted NCI-H1975 cells. RNA sequencing in conjunction with CUT&RUN revealed KDM6A target genes involved in cell cycle regulation, DNA replication, and DNA damage repair pathways, which were further validated using RT-qPCR and CUT&RUN-qPCR. Immunofluorescence confirmed the effects of KDM6A loss on DNA replication and DNA damage-induced repair mechanisms. These findings support our hypothesis that KDM6A is a highly pleiotropic factor in tumorigenesis, playing a critical role in regulating DNA replication-associated gene activation and facilitating the repair of replication stress-induced DNA damage. Notably, loss of KDM6A induced hyperactivation of checkpoint signaling pathways such as the ATR-CHK1 axis, suggesting KDM6A-depleted tumor cells attemption to alleviate replication stress to avoid apoptosis. To evaluate the therapeutic potential of KDM6A along with inhibition of the KDM6A-depletion-induced ATR-CHK1 pathway, ceralasertib (formerly AZD6738), an oral selective ATR inhibitor, was combined with KDM6A depletion for in vivo evaluation. Introducing KDM6A depletion significantly reduced NCI-H1975 tumor volume compared to the control group, demonstrating anti-tumor activity. Consistent with in vitro cell-based assays, the combination therapy of ceralasertib with KDM6A depletion showed enhanced tumor growth inhibition and regression in the NCI-H1975 xenograft model. Our findings reveal a pro-oncogenic role of KDM6A in sustaining tumor maintenance through engagement in DNA replication and repair pathways. Targeting KDM6A alone emerges as a promising therapeutic strategy for NSCLC. Furthermore, combining KDM6A depletion with ceralasertib significantly enhances anti-tumor activity by disrupting the ATR-CHK1 checkpoint signaling pathway, leading to an enhanced anti-tumor activity.Citation Format:Pony Yu-Ling Lee, Kun-Yuan Lin, Chao-Di Chang, Shan-Yun Cheng, Ya-Wen Hung, Chih-Chieh Yang, Yu-Hsien Chang, Chien-Chang Shen, Shu-Ping Wang. Targeting histone H3K27 demethylase for anti-tumor activity: Monotherapy and combination with ceralasertib in non-small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1671.

2024-08-10·Journal of Clinical Oncology

Investigation of a tamoxifen-RACK7/KDM5C-IFN-I axis for ER⁺ breast cancer immunomodulation.

作者: Wang, Shu-Ping ; Guha, Chandan ; Chang, Yao-Ming ; Singh, Saurabh ; Lee, Yu-Ling ; Aberin, Marvin Angelo Esteban ; Lin, Kun-Yuan

9 Background: ER+ breast cancer patients generally have good prognosis. However, significant relapse rates (i.e., hormone drugs, Tamoxifen/TMX) and the poor response to immune checkpoint blockade (ICB) remain critical issues to be addressed. Thus, exploration of anti-ICB resistant mechanisms and alternative targets is needed to improve therapeutic efficacy. Methods: To investigate potential cross-resistant mechanisms between hormone and ICB therapy, ER+ cell lines T47D and MCF7 cells were chronically-treated with TMX for 6 months, and subjected to RNA-Seq and pathway enrichment analysis. Dysregulation of identified factors were validated via western blotting, RT-qPCR, and flow cytometry. Target factors were then inhibited or depleted by shRNA knockdown (KD) or small-compound inhibition. Identified ICB resistance mechanisms were further elaborated by in vitro co-culture assays, syngeneic mouse models, and immune-profiling of tumor microenvironment (TME). Results: Chronic exposure to TMX activates Type I interferon (IFN-I) signaling, induces IFN I-stimulated gene (ISG) expression, and downregulates the H3K4 demethylase and ISG repressor complex known as RACK7/KDM5C. Chronic TMX also upregulates CEACAM1, a well-known ligand for the immune checkpoint receptor TIM3. Results prompt us to evaluate whether loss of RACK7 combined with TMX treatment may modulate a lymphocyte-attractive (via IFN-I), but T-cell exhaustive (via CEACAM1-TIM3) TME. Indeed, in vitro treatment of TMX under RACK7-KD conditions triggers STING upregulation, TBK1 hyperphosphorylation, and a more pronounced activation of ISGs and CEACAM1. In vivo, TMX combined with RACK7-KD enhances tumor growth in the TS/A (ER+) syngeneic mouse model, while immune-profiling reveal that this combination promotes lymphocyte infiltration with a higher population of terminally exhausted CD8+ T-cells in the TME. Conclusions: Our data demonstrate that the TMX-induced activation of cGAS/STING pathway and RACK7 downregulation coordinately shape the IFN-I and immune checkpoint signaling axes in ER+ breast cancer. This TMX–RACK7–IFN-I regulatory network promotes a lymphocyte-attractive TME via IFN-I signaling activation, whereas induction of inhibitory ligands renders T-cells to be terminally-exhausted and unable to kill tumors. These findings highlight the potential use of TMX’s STING-agonistic effect in re-shaping the “cold” breast cancer TME, and the use of combined TMX and anti-TIM3 or anti-CEACAM1 blockade to improve ICB therapy.

2024-06-01·Journal of Clinical Oncology

Depletion of histone H3K27 demethylase exerts anti-tumor activity as a monotherapy and in combination therapy with ceralasertib in non-small cell lung carcinoma.

作者: Wang, Shu-Ping ; Aberin, Marvin Angelo Esteban ; Yeh, Lin-Wen ; Shen, Chien-Chang ; Hung, Ya-Wen ; Chang, Chao-Di ; Lin, Kun-Yuan ; Lee, Pony Yu-Ling ; Chang, Yu-Hsien ; Cheng, Shan-Yun ; Yang, Chih-Chieh

e20035 Background: Lung cancer is one of the most frequently diagnosed cancers and is the leading cause of cancer-related death worldwide. Non-small cell lung carcinoma (NSCLC) represents 85% of lung cancers and is the most common type with poor prognosis. The lysine-specific demethylase UTX (gene name KDM6A) demethylates H3K27me2/3 at genes and enhancers and have been shown to support oncogenic transcription factors. We hypothesize that UTX is a highly pleiotropic factor in tumorigenesis and may plays a critical role in controlling DNA replication-associated gene activation as well as in replication stress-related DNA damage repair. Methods: To evaluate the role of UTX in tumorigenesis, shRNA-mediated UTX knockdown was employed in NCI-H1975 cells followed by cell cycle and cell proliferation assays. In the UTX-knocked down NCI-H1975 cells, target genes involved in DNA replication and DNA damage repair were discovered by RNA sequencing and further validated with RT-qPCR. Effects of UTX depletion on DNA replication and radiation-induced DNA repair were analyzed by immunofluorescence. Effects of UTX depletion on cell cycle arrest and the ATR–CHK1 checkpoint signaling pathway were analyzed by flow cytometry and immunoblotting. In vivo response to UTX inhibition monotherapy (UTX knockdown) and combination therapy of UTX knockdown with ceralasertib, a selective ATR kinase inhibitor, was evaluated in dox-inducible UTX knockdown NCI-H1975 xenografts. Results: Induction of UTX knockdown significantly reduced the NCI-H1975 tumor volume compared to the control group, showing anti-tumor activity. Furthermore, loss of UTX induced hyperactivation of ATR–CHK1 checkpoint signaling pathway, suggesting the UTX-depleted tumor cells attempt to alleviate replication stress to escape from apoptosis. To inhibit the UTX-depletion induced ATR–CHK1 pathway, ceralasertib (formerly AZD6738), an oral potent selective inhibitor of ATR, was combined with UTX-knockdown for in vitro and in vivo evaluation. Consistent with in vitro cell-based assay, combination therapy of ceralasertib with UTX depletion showed enhanced tumor growth inhibition and regression in the NCI-H1975 xenograft model. Conclusions: Our findings provide new insights into a pro-oncogenic role of UTX in supporting tumor maintenance via engaging DNA replication and repair pathway. Depletion of UTX alone may serve as a new therapeutic target in non-small cell lung carcinoma; and further combination with ceralasertib enhances the anti-tumor activity of UTX knockdown by inhibiting the ATR–CHK1 checkpoint signaling pathway.

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