Abstract 7544: Bromodomain and extra-terminal protein inhibitors deplete nuclear factor 1C and reduce stemness and growth of human small cell lung cancer cells in vitro and in vivo
Authors: Wang Haitao , Wang Ruihong , Villa-Hernandez Frank , Shukla Vivek , Varticovski Lyuba , Baek Songjoon , Echtenkamp Frank , Gara Sudheer Kumar , Zhang Mary R. , Shiffka Stephanie , Raziuddin Razi , Neckers Leonard M. , Linehan W. Marston , Hager Gordon L. , Carr Shamus R. , Hoang Chuong D. , Chen Haobin , Schrump David S. 
Published: March 2024

Objectives: Recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential of lung cancers have yet to be fully translated to effective regimens for the treatment of these malignancies. This study sought to examine complex epigenetic mechanisms underpinning the aggressiveness of SCLC, with a focus on understanding those mediating stemness in these highly lethal cancers.

Methods: Normal small airway epithelial cells (SAECs) were reprogrammed into iPSCs, validated by various assays, such as spectral karyotyping and teratoma formation. Genome-wide chromatin landscapes of lung iPSCs (Lu-iPSC) and SAECs, as well as NSCLC and SCLC lines were characterized using RNA-seq, DNA methylome, and DNase-hypersensitivity sequencing (DHS). Gene knock-downs were established using siRNA/shRNA techniques, along with qRT-PCR, immunoblotting, proliferation, and tumorigenicity assays. Glycolysis was analyzed using Seahorse assays. In-silico analysis of the NFIC promoter was performed using TFmapper. NFIC and related gene expression in human SCLC cells following NFIC or Bromodomain-containing protein (BRD) knockdown or exposure to BET inhibitors (BETi) were evaluated. Stemness scores were calculated using established RNA-seq criteria, and gene set enrichment analyses were conducted. Proliferation assays and xenograft models were used for in-vitro and in-vivo SCLC growth assessment.

Results: RNA-seq,DNA methylome and DHS analyses revealed shared characteristics among SAEC, Lu-iPSC, SCLC, and NSCLC, whereas SOXs and POUs showed strong activity in Lu-iPSC but not SCLC. NFIC, with elevated chromatin occupancy in SCLC, exhibited increased expression compared to SAEC. NFIC KD in SCLC significantly curtailed cell growth in-vitro and in-vivo, affecting multiple pathways, including glycolysis and stem cell signaling etc, confirmed by Seahorse assays. Furthermore, we identified occupancy sites for BRD4 in the NFIC promoter, and knockdown of BRD4, but not BRD2 or BRD3, reduced NFIC expression in SCLC cells. BET inhibitor (BETi) treatment dose-dependently decreased NFIC expression in SCLC lines and human SCLC PDXs, irrespective of subtype. BETi treatment also reduced SCLC stemness and showed a significant overlap in gene regulation with NFIC knockdown, affecting pathways related to gluconeogenesis, neuronal differentiation, and embryonic organ morphogenesis. Importantly, BETi treatment effectively reduced SCLC cell growth in-vitro and in-vivo, suggesting the potential therapeutic value of targeting NFIC via BET inhibition in SCLC.

Conclusions: NFIC, a unique transcription factor in SCLC, emerges as a key downstream target of BRD4. These findings underscore the potential druggability of NFIC in clinical settings, provide compelling rationale for evaluating BETi for SCLC therapy.

Citation Format: Haitao Wang, Ruihong Wang, Frank Villa-Hernandez, Vivek Shukla, Lyuba Varticovski, Songjoon Baek, Frank Echtenkamp, Sudheer Kumar Gara, Mary R. Zhang, Stephanie Shiffka, Razi Raziuddin, Leonard M. Neckers, W. Marston Linehan, Gordon L. Hager, Shamus R. Carr, Chuong D. Hoang, Haobin Chen, David S. Schrump. Bromodomain and extra-terminal protein inhibitors deplete nuclear factor 1C and reduce stemness and growth of human small cell lung cancer cells in vitro and in vivo [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 7544.