<i>CXCR4 S338X</i> Promotes Resistance to Second Generation BTK Inhibitor and BCL-2 Inhibitor in WM Cells

Introduction: Waldenström macroglobulinemia (WM) is an IgM-secreting lymphoplasmacytic lymphoma. CXCR4 somatic mutations are common in WM and are associated with clinical resistance to Bruton's tyrosine kinase (BTK) inhibitors and other therapeutic agents. CXCR4 mutations occur nearly exclusively with MYD88 L265Pmutation, with CXCR4 S338X constituting the most common in WM patients. The aim of this study was to further investigate the effect of CXCR4 S338X on BTK inhibitors (ibrutinib and zanubrutinib) and a BCL-2 inhibitor (venetoclax) resistance. Methods: MWCL-1 cells were transfected with scramble, CXCR4 or CXCR4 S338Xplasmids using Lipofectamine 3000 followed by neomycin selection. CXCR4 expression was confirmed using qPCR and flow cytometry. Transfected cells were cultivated in presence of ibrutinib (5μΜ), zanubrutinib (5μΜ) and venetoclax (1μΜ) for 48 hours. Proliferation was estimated using CCK-8 colorimetric assay while cell cycle was evaluated by propidium iodide staining and flow cytometry. Apoptosis was estimated by Annexin V/7-AAD staining and flow cytometry while the expression of apoptotic genes was determined by qPCR. The statistical analysis was performed using Student's t-test and Graphpad Prism. Results: Drug-induced cytotoxicity was observed only in the case of venetoclax in CXCR4 S338X transfectedcells (p= 0.0007) while increased cytotoxicity was noted in the presence of ibrutinib, zanubrutinib and venetoclax in the case of scramble-transfected cells (p=0.038, p=0.006 and p=0.0006, respectively). Moreover, no significant difference in proliferation rate was noted between CXCR4 S338Xtransfectedcells treated with the therapeutic agents and CXCR4 S338X untreated cells, underlining the S338X role to promote cell proliferation. Interestingly, CXCR4 S338X transfectedcells exhibited apoptosis resistance not only compared to scramble-transfected cells but also in comparison with CXCR4-transfected cells, associated with resistance to all three drug agents (p=0.004, p=0.003 and p=0.034, respectively). In the case of scramble-transfected cells, an increase in apoptosis was demonstrated in parallel with an increase in caspases 8, 9 and 3 in presence of ibrutinib (p=0.006, p=0.0001 and p=0.025, respectively) and zanubrutinib (p=0.027, p=0.007 and p=0.052, respectively), whereas no statistically significant increase in caspase gene expression was observed in the case of CXCR4 S338Xtransfectedcells, highlighting S338X role in the inhibition of intrinsic and extrinsic apoptosis pathways. Lastly, the presence of S338X mutation acted as a mitotic promoting factor when cells treated with the aforementioned drugs showed a high proportion of cells in G2/M phases of cell cycle compared to scramble and CXCR4 controls. Conclusions: The above data demonstrate the role of CXCR4 S338X mutation in drug resistance and suggest the necessity to develop therapeutic strategies to address this issue and overcome this therapeutic barrier in WM. Data on more cell lines and downstream signaling will be presented at the meeting.