Single cell multiomics dissection of In Vivo immune modulation and reconstitution induced by bruton's kinase inhibitors in CLL patients

Abstract Background: Bruton's Tyrosine Kinase inhibitors (BTKi) have transformed the treatment landscape of Chronic Lymphocytic Leukemia (CLL). In addition to their direct anti-tumor effect, these targeted drugs have demonstrated a unique ability to modulate the tumor microenvironment. These effects, as well as the differential impact of various types of BTKis and their impact on outcome remain incompletely understood. Aim: To study the cellular and molecular mechanisms of immune modulation and reactivation during treatment with either Ibrutinib+Venetoclax (I+Ven) or acalabrutinib (A) in patients with CLL. Methods: We performed a longitudinal single-cell RNA sequencing of the peripheral blood (PB) of CLL patients before and during treatment with BTKis at 2 weeks, 3 and 12 months. Results: Thirty-five treatment naïve patients with CLL were enrolled (n, I+Ven= 17 ; A= 18). Patients in the I+Ven arm received treatment for a fixed duration, whereas patients in the A arm were treated with continuous single-agent therapy. Median age was 75.5 (Interquartile range, 66-78), 25 patients were IGHV unmutated. At the onset of treatment, Binet stages were distributed as follows: 13 stage A, 13 stage B, 9 stage C. Ten patients experienced infectious complications. Response was defined according to the IWCLL 2018 general practice criteria. Seventeen patients achieved Complete Remission (CR) at 12 months, while 18 patients achieved Partial Response (PR). We profiled 134,896 high quality cells ( 34,795 CLL, 100,101 Immune) from 18 CLL patients (n: I+Ven= 11, A=7) across four time points during treatment and 10 healthy controls (HC). Prior to treatment, in comparison to healthy controls, CLL patients exhibited T-cell dysregulation marked by loss of naïve and memory subsets, increased apoptotic signaling, and impaired metabolic (PI3K-AKT), antigen processing (KEGG Proteasome), and type I interferon genomic pathways. In the myeloid compartment, classical and non-classical monocytes showed innate immune suppression via FcγR pathway downregulation, while intermediate monocytes preserved FcγR activity and upregulated inflammatory genes, indicating retained immune functionality. Distinct immune remodeling patterns were associated with response to BTKi in a treatment specific manner with substantial interpatient variability. Expansion of the myeloid compartment correlated with better outcomes, especially in patients treated with A. Specifically, A-treated patients who achieved CR showed enrichment of conventional dendritic cells and Central Memory T cells, whereas patients at PR demonstrated depletion of Non-Classical (CD16+) monocytes and enrichment of intermediate monocytes (CD16+CD14+). In I+Ven treated CR patients, we observed an enrichment of intermediate monocytes alongside a reduction in terminal effector T cells and central memory T cells. Patients in PR under I+Ven exhibited an expansion of terminal effector T cells. Addition of Ven to I at 3 months on treatment resulted in a downregulation of the bcl2 pathway in both T and myeloid cells at the 12 months time point, and was positively correlated with complete response. During treatment, monocyte subsets underwent distinct transcriptional changes associated with clinical response. In CR patients, Classical (CD14⁺) monocytes shifted toward a favorable pro-inflammatory state with increased IL-12 signaling and reduced interferon- α response, suggesting enhanced T/NK cell activation. Intermediate monocytes in CR patients showed sustained immune activation and survival signaling, with upregulation of TNF/NF-κB, IL-12, IFN- α, and IFN- β pathways, while Non-classical monocytes remained transcriptionally unchanged, retaining a metabolically suppressed and pro-apoptotic profile. Data on the microenvironment of additional 16 patients will be presented at the ASH. Conclusions: BTKi treatment induces distinct transcriptional changes across T and monocyte subsets, and responders exhibit a restoration of pro-inflammatory and antigen-presenting functions. Preliminary results suggest that each BTKi induces distinct immune signatures, with potential implications on combinations therapies, specifically with Ven.These differences could impact tumor clearance and the durability of remission, carrying important implications for time-limited combination strategies in CLL.