Background: Mutations in the Toll receptor (TLR) pathway are highly prevalent in Waldenstrom's Macroglobulinemia (WM) and ABC DLBCL, wherein mutated MYD88 triggers NF-kB pro-survival signaling through BTK/IRAK. Activation of B-cell receptor (BCR) signaling can also be triggered by activating mutations in CD79A/B in ABC DLBCL, though are rare in WM (5-8%). Despite these findings, there is evidence for BCR activation in WM (Argyropoulos et al, Leukemia 2016). We hypothesized that crosstalk between TLR and BCR might account for aberrant BCR signaling in WM. We therefore performed PhosFLow studies on MYD88 and BCR signaling components and observed the hyperactivation of SYK (pY525-pY526) in MYD88 mutated WM (BCWM.1, MWCL-1) cells compared to wild-type MYD88 expressing control cells. We next treated MYD88 mutated WM cells with a peptide inhibitor of MYD88 homodimerization, and observed that the phosphorylation of SYK at tyrosine 525 and 526 (pY525-pY526) together with phosphorylation of BTK at tyrosine 223 (pY223) was markedly reduced. In contrast, the MYD88 inhibitor had no impact on either SYK or BTK activation in MYD88 wild-type Ramos cells. Knockdown of MYD88 by lentiviral transduction in MYD88 mutated BCWM.1 cells confirmed the reduction of SYK (pY525-pY526) by two shRNAs. Cell viability analysis by CellTiter-Glo® assay showed that the SYK inhibitor R406 was cytotoxic to MYD88 mutated WM cells at pharmacologically attainable drug levels (EC50 0.247 uM for BCWM.1; 0.315 uM for MWCL-1), versus >1 uM for MYD88 wild-type control cells. Furthermore, the combination of R406 with ibrutinib produced synergistic cytotoxicity in MYD88 mutated WM cells. Conclusion: Our findings demonstrate that in addition to TLR path activation, mutated MYD88 activates the BCR component SYK tyrosine kinase. These findings provide the rationale for combined therapeutic targeting of BCR and TLR pathways in MYD88 mutated WM.
Castillo: Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Research Funding. Treon: Pharmacyclics: Consultancy, Research Funding.
Asterisk with author names denotes non-ASH members.