Abstract 3273: BTK inhibitors reshape tumor transcriptome and lymph node microenvironment in CLL

Abstract Bruton tyrosine kinase inhibitors (BTKis) have transformed the treatment landscape for patients with chronic lymphocytic leukemia (CLL). BTKis target pathways critical for CLL cell survival and proliferation. We evaluated the effects of BTKis on CLL cell transcriptome and the composition of the tumor microenvironment (TME) in CLL. Lymph node (LN) biopsies from 65 patients treated with ibrutinib (n=35) collected pre-treatment and at 24 and 72 hours from first dose or acalabrutinib (n=30) collected pre-treatment and after 3 days of treatment at 4, 12, 24, and 36 hours from the last dose of drug were submitted for RNA sequencing. We used gene set enrichment analysis (GSEA) to interrogate activation of signaling pathways and cellular response and quantified the degree of CLL activation using a previously published gene signature derived from single-cell RNA sequencing. Immune cell composition was assessed with CIBERSORT. GSEA highlighted significant downregulation of key CLL-related pathways by BTKis, including MYC, KRAS, HIF-1, NOTCH, NF-κB, and BCR, all with adjusted p = 0.007. Consistently, the CLL activation gene signature showed a significant decrease on BTKis (p = 0.015). To better understand the timing of decreased activation, we analyzed ibrutinib timepoints separately. Activation score decreased by 23% after 24 hours (p = 0.24) and by 87% after 72 hours of ibrutinib (p = 0.014). We then investigated the acalabrutinib timepoints: the activation score decreased by 73.7% at 4 hours (p = 0.013), 61.7% at 12 hours (p = 0.016), 101.6% at 24 hours (p = 0.056). How BTKis affect the LN TME is not well known. The LN TME in CLL differs from normal LNs in several respects, including increased presence of T follicular helper (Tfh) cells, and decreased presence of CD4+ naïve T cells and M0 macrophages. In ibrutinib treated LN biopsies, CIBERSORT identified a significant decrease of Tfh after 72 hours (p=0.001) but not after 24 hours, while the proportion of resting CD4 memory T cells increased (p = 0.003). LN biopsies taken >72 hours after the start of acalabrutinib revealed significant decreases in Tfh cells (p = 0.00003), CD4 memory activated T cells (p = 0.03), M0 macrophages (p = 0.028), and increases in CD4 naïve T cells (p = 0.018), CD4 memory resting T cells (p = 0.012), M2 macrophages (p = 0.03), and resting mast cells (p = 0.0097). In conclusion, BTKis not only rapidly reduced activating signaling in CLL cells but also significantly altered the composition of the LN TME. The virtually immediate impact on tumor transcriptomes was paralleled by equally rapid if slightly delayed reorganization of the TME. These data suggest that the effects of BTKis spread beyond the immediate drug target and disrupt the tumor-supportive TME. Citation Format: Victoria M. Rogness, Rohan Herur, Bharath Dileep Kumar, Christine Gruessner, Clare Sun, Chingiz Underbayev, Adrian Wiestner. BTK inhibitors reshape tumor transcriptome and lymph node microenvironment in CLL [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 3273.