The Combined Treatment of Btkis with Venetoclax Exhibits an Enhanced Therapeutic Effect on Waldenström Macroglobulinemia Cells Bearing CXCR4 Mutations

Waldenström macroglobulinemia (WM) is an IgM-secreting lymphoplasmacytic lymphoma characterized by recurrent somatic mutations in CXCR4 gene that are present in about 30-40% of WM patients always co-oocur with MYD88L265P mutation, and are associated with clinical resistance to therapeutic agents including Bruton's tyrosine kinase inhibitors (BTKIs). The aim of this study was to investigate the therapeutic effect of the combination of BTKIs, ibrutinib and 2nd generation zanubrutinib, with a BCL-2 inhibitor (venetoclax) in the CXCR4 mutated (CXCR4MUT)cells. MWCL-1, BCWM-1 and OCI-LY3 cells were transfected with scramble, CXCR4S338X, CXCR4R334X, CXCR4G336X, CXCR4E434K or CXCR4E343X plasmids using Lipofectamine 3000. CXCR4 expression was determined using qPCR and flow cytometry (FC). Transfected cells were cultured in presence of ibrutinib (5μΜ), zanubrutinib (5μΜ), venetoclax (1μΜ) or the combination of BTKIs with venetoclax for 48 hours. Proliferation was estimated using CCK-8 while cell cycle was evaluated by propidium iodide staining (FC). Apoptosis was estimated by Annexin V/7-AAD staining (FC) while the expression of apoptotic genes was determined by qPCR. RNA-seq analysis was performed in MWCL-1 and BCWM-1 cells transfected with CXCR4MUT plasmids or wild-type/scramble with or without BTKIs, venetoclax or their combination. Our results showed that all 5 CXCR4MUT cells were rescued from drug-induced cytotoxicity in the cases of treatment with single therapeutic agents. The combination of BTKIs with venetoclax led to decreased CXCR4 expression and statistically increased apoptosis on both MWCL-1 (p=0.003 for CXCR4R334X, p=0.002 for CXCR4G336X and CXCR4E343X, p=0.001 for CXCR4E343K) and BCWM-1 cells (p=0.001 for CXCR4R334X and CXCR4G336X, p=0.002 for CXCR4E343X) compared with untreated CXCR4MUT cells. Moreover, we observed statistically significant decreased proliferation in the cases of combined treatment (P ≤ 0.001 for all CXCR4MUT cells) compared with monotherapies leading to cell cycle arrest and eliminating the percentage of cells undergoing G2/M phase. Preliminary data on OCI-LY3 cells, which is homozygous to MYD88L265P, showed that the transfection with CXCR4MUT plasmids also triggered resistance to BTKIs treatment, whereas the combination treatment of BTKIs with venetoclax led to reduced cell survival (82.2 ± 16.9%) and decreased proliferation ratio (0.4 ± 0.17) compared with untreated cells (2.28 ± 0.67). When comparing data from zanubrutinib to ibrutinib either as a single agent or in combination with venetoclax, we observed a statistically higher cytotoxicity (P = 0.033 for CXCR4S338X, P = 0.03 for CXCR4E343Kin BCWM-1 cells and P = 0.014 for CXCR4S338X in MWCL-1 cells) and lower cell proliferation (P ≤ 0.01 for all CXCR4MUTcells). RNA-seq analysis of treated versus all untreated cell lines bearing any of the tested CXCR4 mutations (n=72), showed that the molecular mechanism governing the drug resistance of CXCR4MUT cells was mainly driven by the upregulation of genes involved in AKT, RAS and NFkB signaling. Gene enrichment analysis of BTKIs monotherapies showed lower expression of genes involved in signal transduction by growth factors such as GR2, MAP2K, AC015871, LYN, DNMT1, SMAD3/4, G2/M transition such as CDK-1 and ALMS1 and MAPK signaling such as FGB, MARK2 and MAP2K2 in wild type/scrambled transfected cells, while in the case of CXCR4MUT cells only the reduction of BLNK1 gene (acting on NFkB signaling) was observed. Lastly, the combination treatment, for both ibrutinib and zanubrutinib, resulted in decreased expression of genes involved in BCR and NFkB signaling such as HRAS, IKBKG, BANK1, CD81, NFKB1, FCGR2B, MAPK1, IRAK4 and PIK3R3 as well as increased expression of apoptotic genes such as CASP3, CASP9 and HRK, in both CXCR4WT and CXCR4MUT cells. Overall, our data show the synergistic action of BTKIs with venetoclax not only in CXCR4WT cells but also in CXCR4MUTcells, that may also exhibit an enhanced therapeutic benefit overcoming the CXCR4MUT- induced drug resistance seen in patients with WM.