Evolution of tumor subclones and T-cell dynamics underlie variable ibrutinib responses in Waldenström macroglobulinemia

To elucidate the molecular basis underlying differential response and resistance to ibrutinib in Waldenström's macroglobulinemia (WM), we conducted a prospective phase II trial (ClinicalTrials.gov; NCT02604511) of ibrutinib monotherapy in treatment-naïve patients. Seventy-four sequential bone marrow (BM) aspirates from 17 patients, collected from baseline through 48 treatment cycles, were profiled using single-cell multi-omics. BM cells segregated primarily into B/plasma cell and T-cell compartments. Longitudinal clonal tracking of malignant B/plasma cells identified three distinct evolutionary patterns: "evolution" (early clone contraction with late clone expansion and increasing genomic complexity), "devolution" (early clone expansion with late clone contraction and genomic simplification), and "no-evolution" (stable clonal architecture). The "evolution" pattern was strongly associated with disease progression, whereas "devolution" correlated with durable clinical response. Transcriptomic profiling of resistant clones enabled development and validation of the Waldenström's Ibrutinib Prediction (WIP) score, which predicted treatment response at baseline. Within the WIP signature, LYN emerged as a key regulator; LYN knockdown or inhibition significantly increased WM cell sensitivity to ibrutinib, suggesting a rational combinatorial strategy. In parallel, GZMB⁺ CD8⁺ effector-memory (TEM) cells expanded post-treatment in progressing patients and co-existed with tumor "evolution". These cells exhibited persistently impaired cytotoxic programs (e.g., GNLY), a de-differentiated memory-like state, elevated PDCD1 expression, and reduced TCR diversity. Together, this study provides the first single-cell framework of tumor clonal evolution and T-cell dysfunction under ibrutinib in WM; introduces the WIP score as a predictive biomarker for treatment response; and identifies actionable tumor-intrinsic and immune mechanisms driving resistance.