Abstract

Abstract 897

Background:

MYD88 is an adapter molecule which promotes Toll-Like Receptor and IL-1 Receptor signaling through the IRAK 1/4/TRAF6/NF-κB pathway. MYD88 L265P is a recently described somatic mutation that is widely expressed (>90%) in patients with Waldenstrom's Macroglobulinemia (Treon et al, NEJM 2012). The signaling pathway by which MYD88 L265P promotes WM growth and survival remains to be fully clarified. We therefore sought to identify pathway(s) responsible for MYD88 L265P pro-survival signaling.

Patients and Methods:

Primary WM-LPCs were isolated from bone marrow biopsy specimens from WM patients. Normal CD19+ B-cells were isolated from the peripheral blood of healthy donors. Western blot analyses was performed using total and phospho-specific antibodies in the MYD88 L265P expressing WM cell lines BCWM.1 and MCWL-1 following MYD88 knockdown by lentiviral transduction, and/or use of MYD88 or IRAK signal inhibitors. WM cells were also treated with the BTK inhibitor PCI-32765, in the presence or absence of an IRAK1/4 inhibitor. Cell survival was evaluated by Annexin V/PI staining and/or AlamarBlue® or ATPLite® assays after treatments with inhibitors. Synergistic interactions were assessed with CalcuSyn software.

Results:

Lentiviral knockdown of MYD88 and/or use of an inhibitor of MYD88 homo-dimerization led to decreased survival of MYD88 L265P expressing BCWM.1 and MWCL-1 cells, as well as primary WM LPCs expressing MYD88 L265P. In contrast minimal killing was observed in MYD88 Wild Type (WT) expressing Ramos, MM.1S, or healthy donor CD19+ selected B-cells. Phospho-specific western blot analysis showed decreased IRAK1, NF-kB and STAT3 signaling following inhibition of MYD88 signaling in L265P expressing WM cells. Conversely, use of an IRAK1/4 kinase inhibitor showed reduced tumor cell killing, NF-kB and STAT3 signaling. We therefore performed co-immunopreciptation experiments to identify other MYD88 binding partners in L265P expressing WM cells. These studies identified Bruton's Tyrosine Kinase (BTK) in complex with MYD88, with MYD88 showing more robust binding to phospho-BTK in L265P expressing WM cells. Lentiviral knockdown of MYD88 and/or use of an inhibitor of MYD88 homodimerization led to decreased BTK phosphorylation. Moreover, overexpression by lentiviral transduction of MYD88 L265P showed more robust BTK phosphorylation versus WM cells transduced with MYD88 WT. Lastly, the use of a BTK-inhibitor (PCI-32765) blocked NF-kB and STAT3 signaling, as well as induced WM tumor cell killing that was synergistically enhanced by the presence of an IRAK1/4 kinase inhibitor.

Conclusion:

MYD88 L265P promotes survival of WM cells by activation of BTK. These studies provide the framework for the investigation of BTK inhibitors in WM, as single agents and in combination with other MYD88 pathway inhibitors.

Disclosures:

Treon:Onyx: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Cephalon: Consultancy; Avila: Consultancy.

Author notes

*

Asterisk with author names denotes non-ASH members.