TENT5C As a Target of Chromatin Remodeler SMARCA2 Participates BTK Inhibitor Resistance in Mantle Cell Lymphoma By Regulating ATP Metabolism

Background: Mantle cell lymphoma (MCL) is an incurable lymphoma with high clinical heterogeneity. The application of BTK inhibitors (BTKi) has improved the prognosis of MCL patients, but most patients still experience disease progression within a few years, and those with relapsed MCL respond poorly to available salvage therapies. Elucidating the mechanisms of MCL resistance, early identification of resistant populations, and exploring targetable key genes and combination therapy strategies are crucial for improving the prognosis of such patients.

Methods: In this study, we constructed stable cell lines with SMARCA2 knockdown and overexpression (Jeko-1 SMARCA2^sh, Jeko-1 SMARCA2^OE, Z138 SMARCA2^sh, Mino SMARCA2^sh) and induced ibrutinib-resistant cell lines (Z138-R, Z138-R SMARCA2^OE). We verified the effects of SMARCA2 on cell proliferation, cell cycle, pathway abnormalities, and sensitivity to ibrutinib in vitro and in vivo. Transcriptome and ATAC-sequencing were performed with SMARCA2^sh cell lines before and after treated with ibrutinib. The evaluation of mitochondrial function was achieved by detecting ATP release, oxygen consumption, mitochondrial membrane potential, and ROS production in SMARCA2^sh and SMARCA2^OE cells. The anti-MCL activity and mechanism of Isoginkgetin (IGK) monotherapy or combination with Ibrutinib were evaluated in SMARCA2^sh or Ibrutinib resistant MCL cells in vitro and in vivo.

Results: Preliminary studies [JCI 2022, doi: 10.1172/JCI153283] involving WES analysis of 134 MCL patient samples and RNA sequencing of 62 MCL patient samples found that SMARCA2 deletion or low expression was associated with MCL progression, BTKi resistance, and shorter patient survival. In vitro and in vivo experiments confirmed that SMARCA2 downregulation promoted MCL cell proliferation, extended the S phase of the cell cycle, and reduced sensitivity to ibrutinib. SMARCA2^sh cells exhibited elevated ATP levels, increased oxygen consumption, and downregulated ROS levels. Ibrutinib inhibited ROS levels, but its inhibitory effect was diminished in SMARCA2^sh cell lines, leading to Ibrutinib resistance. Patient-derived primary cell experiments confirmed that the inhibitory effect of ibrutinib on ROS was weakened in patients with SMARCA2 deletion.

Joint analysis of RNA-seq and ATAC-seq data from cell lines with SMARCA2^sh and RNA-seq from patient samples indicated that the pathway which SMARCA2 regulated were enriched in ROS production, ATP metabolism, and mitochondrial inner membrane-related pathways. Additionally, SMARCA2 downregulation led to decreased the accessibility of the intron regions of TENT5C genes, and higher H3K27me3 suppressive modification for downregulated expression of TENT5C. Overexpression of TENT5C in SMARCA2^sh cell lines led to decreased cell proliferation, reduced ATP levels, upregulated ROS levels, and restored sensitivity to ibrutinib. These results were also validated by knocking down TENT5C in SMARCA2^OE cell lines. Multicolor immunohistochemistry in tumor issue from mouse model and immunofluorescence assays of MCL cell line revealed co-localization of TENT5C/FAM46C with ATP5A and SLC25A5 in mitochondrial. Co-immunoprecipitation experiments further confirmed that FAM46C may regulate ATP synthesis and ADP/ATP transformation to affect drug resistance by interacting with ATP5A and SLC25A5 respectively.

IGK has been reported to be effective in FAM46C knockout myeloma cell lines. Our in vitro experiments demonstrated the synergistic therapeutic effect of IGK and ibrutinib. Mechanistically, IGK inhibited ATP levels, restored ROS production, activated the BCR signaling pathway, thereby reversed ibrutinib resistance in SMARCA2^sh and Z138-R cell lines. Finally, we used Cell Line-Derived Xenograft (CDX) and Patient-Derived Xenografts (PDX) models to further validate the synergistic effect of IGK and ibrutinib.

Conclusion: Our study first highlights that SMARCA2 regulates the ATP metabolism by altering the accessibility and expression of the TENT5C gene, thereby affecting MCL proliferation and BTKi resistance. IGK enhances ibrutinib efficacy by reducing ATP production, and exhibits a synergistic effect with ibrutinib. These findings provide novel therapeutic targets and strategies to overcome BTK inhibitor resistance in MCL.