Participation of BTK in MYD88 signaling in malignant cells expressing the L265P mutation in Waldenstrom’s macroglobulinemia, and effect on tumor cells with BTK-inhibitor PCI-32765 in combination with MYD88 pathway inhibitors.

Abstract

8106 Background: Bruton’s tyrosine kinase (BTK) promotes B-cell receptor signaling along with B-cell expansion and survival through NF-κB and MAPK. MYD88 L265P is a widely expressed somatic mutation in tumor cells from WM patients. MYD88 L265P promotes enhanced tumor cell survival through IRAK 1/4 mediated NF-κB and MAPK signaling. We therefore sought to clarify the role of BTK signaling in MYD88 L265P expressing WM cells, and the impact of BTK and MYD88/IRAK inhibition on WM cell signaling and survival. Methods: Western blot analysis was performed using total and phospho-specific BTK antibodies in MYD88 L265P expressing primary WM patient cells, BCWM.1 and MCWL-1 WM cell lines following MYD88 knockdown by lentiviral transduction, and/or use of MYD88 or IRAK signal inhibitors. Cells were also treated with the BTK inhibitor PCI-32765, in the presence or absence of MYD88 homodimerization or IRAK1/4 inhibitors. Annexin V / PI staining was used to assess cell survival, and synergism assessed with CalcuSyn software. Results: BTK was phosphorylated in MYD88 L265P expressing WM cells. Knockdown of MYD88 by lentiviral transduction, and/or use of MYD88 or IRAK 1/4 kinase inhibitors led to decreased BTK phosphorylation. Phosphorylation of BTK, TIRAP, a major TLR adapter protein for MYD88 signaling, IRAK1, IKBα, ERK1/2 and STAT3 were significantly reduced following treatment with PCI-32765. Treatment with PCI-32765 also induced apoptosis of MYD88 L265P expressing WM cells, and showed synergistic tumor cell killing in the presence of either MYD88 homodimerization or IRAK 1/4 kinase inhibitors. Conclusions: BTK activation is facilitated by MYD88 pathway signaling in L265P expressing WM cells, and participates in MYD88 downstream signaling. Inhibition of BTK by PCI-327625 led to robust tumor cell killing of MYD88 L265P expressing WM,cells, which was potentiated by MYD88 pathway inhibitors. These studies provide the framework for the investigation of BTK inhibitors in WM, as single agents and in combination with MYD88 pathway inhibitors.