TLR9-dependent migration identifies different Chronic Lymphocytic Leukemia signaling states under BTK inhibition

B-cell receptor (BCR) inhibitors have transformed chronic lymphocytic leukemia (CLL) therapy, yet many patients show incomplete responses or develop resistance in the absence of Bruton's tyrosine kinase (BTK) mutations. We investigated Toll-like receptor 9 (TLR9) signaling as a tumor-response mechanism to BCR blockade. Using 102 TLR9-stimulated patient samples, we identified two distinct migratory phenotypes: TLR9-induced migratory Responders (R), with enhanced migration, and TLR9-induced migratory Reverse-Responders (RR), with suppression of migration following TLR9 activation. Transcriptomic and protein analyses showed TLR9-stimulated R samples expressed higher IRAK4/IRAK1 and induced NF-kB p100, consistent with intact TLR9 signaling. In contrast, RR samples exhibited elevated basal BCR activity, and lower IRAK4/IRAK1 with rapid IRAK1 degradation, indicating suppressed TLR9 signaling. In-silico modeling and biochemical validation showed high basal BCR signaling suppresses TLR9 responsiveness, whereas low basal activity permits full TLR9 activation. Functionally, TLR9-induced migration persisted despite BTK inhibition (BTKi) in R but not RR samples, and was abrogated by TLR9 antagonism, consistent with signaling through a BTK-independent pathway in the R subgroup. CLL cells from BTKi-resistant patients expressed higher TLR9 and matched pre- and post-relapse samples showed decreased BCR and increased TLR-associated NF-kB signaling post-relapse. Clinically, in small exploratory.