Inhibition of lysine acetyltransferases p300/CBP overcomes BTK inhibitor resistance in a xenograft model of Mantle Cell Lymphoma by suppressing MYC signaling

Abstract

Abstract Background Bruton's tyrosine kinase inhibitors (BTKis) have improved outcomes for patients with relapsed/refractory mantle cell lymphoma (MCL). However, the development of drug resistance remains a major challenge. Using a CRISPR/Cas9 screen, we identified p300/CBP inhibition as a promising strategy to overcome BTKi resistance. p300/CBP function as histone acetyltransferases (HATs), regulating chromatin structure and the transcription of oncogenic drivers such as MYC. Clinical evidence indicates that MYC is frequently upregulated in BTKi- and subsequent chimeric antigen receptor T-cell (CAR-T) therapy-resistant MCL. Preclinical studies have shown that p300/CBP inhibitors (p300/CBPis) can suppress tumor growth and reactivate immune-related genes, and early-phase trials have demonstrated encouraging efficacy and tolerability (NCT03568656, NCT04068597). We aimed to validate the mechanistic basis of the synergy between BTKis and p300/CBPis and to assess the safety and efficacy of this combination in vivo using a BTKi-resistant MCL xenograft model. Methods We treated four MCL cell lines (Granta519, Mino, Jeko-1, and REC1) with ibrutinib, the p300/CBPi A-485, or both for 1–2 weeks. Cell viability was assessed using MTS assay to evaluate the synergistic effects of ibrutinib and A-485. Cell cycle arrest and apoptosis were analyzed by flow cytometry using PI staining and Annexin V/7-AAD assays. To examine downstream signaling, BTK, PLCγ2, and AKT phosphorylation was analyzed by Western blotting. We conducted RNA sequencing (RNA-seq) to profile transcriptional changes following ibrutinib, A-485, or combination treatment, and performed pathway enrichment analysis and GSEA. For in vivo studies, we xenografted 7-week-old female NOG mice with luciferase-labeled Granta519 cells and randomized them into DMSO, ibrutinib, A-485, or combination therapy group (n = 7/group). Therapeutic efficacy was evaluated by measuring tumor burden using calipers and bioluminescence imaging with an In Vivo Imaging System (IVIS), whereas safety was monitored by tracking body weight and general condition. Results During a 1–2-week culture period, the combination of a BTKi and a p300/CBPi demonstrated synergistic suppression of cell proliferation in four MCL cell lines (Granta519, Mino, Jeko-1, and REC1). MTS assay showed a synergistic effect in both BTKi-resistant Granta519 and sensitive cell lines. The p300/CBPi increased ibrutinib sensitivity, leading to up to four-fold greater suppression of cell viability. The synergistic effect of p300/CBPi was greater than when combined with PI3K inhibitor (idelalisib) or BCL2 inhibitor (venetoclax). This combination also promoted G1 cell cycle arrest and apoptosis. Furthermore, the combination reduced AKT and BTK, PLCγ2 phosphorylation, indicating effective BCR signaling inhibition. RNA-seq analysis revealed that the combination treatment most strongly downregulated MYC target pathways (Q value = 0.007). MYC overexpression reduced the efficacy of the combination. BTKi monotherapy (ibrutinib) activated IL-6/JAK/STAT3 signaling, whereas the addition of p300/CBPi (A-485) reversed this activation. Downregulated genes within the IL-6/JAK/STAT3 pathway include CCND1 and CD40, both critical for MCL proliferation. We also observed that BTKi-resistant cells exhibited higher baseline levels of STAT3 phosphorylation than sensitive cells, and that the combination treatment further reduced this phosphorylation. In a xenograft model, the combination therapy significantly suppressed tumor growth compared to vehicle or single-agent treatments. Mean tumor volumes (mm³) at the endpoint were: DMSO, 1314.7; ibrutinib, 949.0; A-485, 1022.8; and combination, 397.2 (all p < 0.001 vs. combination). IVIS imaging confirmed reduced tumor burden, with significantly lower total flux (photons/sec) in the combination group than in the DMSO group (mean 18.8 vs 35.0 × 108 p/s, p = 0.022). No significant weight loss or toxicity was observed in any treatment group. Western blot analysis of harvested xenograft tumors showed that the combination reduced phosphorylation of BTK, AKT, and STAT3, and decreased downstream MYC protein expression. Conclusion p300/CBP inhibition overcomes BTKi resistance in MCL primarily by suppressing MYC-driven signaling. These findings provide strong preclinical support for clinical evaluation of BTKi and p300/CBPi combination therapy in patients with BTKi-resistant MCL.