The effects of bruton tyrosine kinase inhibitors on the waldenstrom macroglobulinemia microenvironment Free

Waldenstrom Macroglobulinemia (WM) is a rare lymphoplasmacytic lymphoma driven primarily by the activating mutation MYD88 (L265P) and characterized by elevated IgM production. Progression from IgM MGUS (Monoclonal Gammopathy of Undetermined Significance) to WM is marked by bone marrow infiltration, where tumor stroma and immune cell interactions play a critical role in disease biology. Cytokine-driven positive feedback loops involving paracrine signaling between malignant cells, stromal fibroblasts, and innate immune cells, contribute to WM cell survival and immune evasion. While WM remains incurable, treatment strategies such as R-CHOP have provided clinical benefit. More recently, Bruton Tyrosine Kinase (BTK) inhibitors have shown promise due to their ability to disrupt B-cell receptor (BCR) signaling, downstream of BTK activation, including pathways such as STAT3, NF-κB, MAPK, and AKT.However, the extent to which BTK inhibitors influence the TME, especially in terms of immune modulation and evasion, remains largely unexplored. Based on this, we hypothesize that BTK inhibitors exert part of their therapeutic effect through modulation of the immune microenvironment.

We investigated how BTK inhibition alters the transcriptional landscape in WM models, specifically focusing on immune signaling and chemotaxis pathways. We treated WM cells lines (BCWM.1, MWCL-1 and RPCI-WM1) with acalabrutinib, a highly selective BTK inhibitor, and probed for broad changes in cytokines/chemokines gene expression through RNA-seq and signaling specific proteomic arrays. We observed a significant reduction in the expression of the chemokines CCL3 and CCL4, which are known to recruit macrophages, in BCWM.1 and MWCL-1 cells, but not in the BTK inhibitor insensitive RPCI-WM1 cells. These transcriptional changes were confirmed by RT-qPCR (24 h post-treatment) and ELISA (72 h post-treatment) using two different BTK inhibitors, acalabrutinib and ibrutinib. Downregulation of CCL3 and CCL4 corresponded with a reduction in BTK activation, measured by decreased phosphorylation at Y223, a critical auto-phosphorylation site required for downstream BTK signaling. Given the established role of CCL3 and CCL4 in macrophage recruitment to the bone marrow in WM, we assessed macrophage migration using a trans-well assay with WM cell-conditioned media. BTK inhibitor-treated conditioned media significantly reduced macrophage (CD14+-derived from PBMCs of healthy donors) migration. We observed upon BTK inhibition, that MEK1/2 signaling is reduced, as evidenced by decreased phosphorylation of MEK1/2 as well as the downstream node of ERK1/2. Mechanistically, the effects observed are linked with the MEK1/2-ERK1/2 axis, as pharmacological inhibition of MEK1/2 downregulated CCL3/CCL4 secretion and reduced macrophage recruitment, mirroring the effects of BTK inhibition.

To further dissect tumor-immune interactions, we co-cultured WM cells with M0 macrophages. Co-culture induced an M2-like phenotype in macrophages over 7 days, evidenced by increased expression of CD163 and CD206 by spectral flow cytometry. RNA-seq of co-cultured WM cells showed up-regulation of MAPK and IL-2/STAT5-associated genes, suggesting bidirectional signaling. Together, these findings reveal that BTK regulates immune-modulating chemokines via ERK signaling and that BTK inhibitors may function in part by disrupting WM-macrophage interactions within the bone marrow microenvironment.