Mechanisms of Suppressive Effects of MYD88 Inhibitors on the Growth of Lymphoma and Leukemia Cells

Introduction : Myeloid differentiation primary response gene 88 (MYD88) is an adaptor protein that binds to the Toll-like/interleukin-1 receptor to form a homodimer and activates NF-kB pathway. Activating MYD88 mutations are found in 90% of lymphoplasmacytic lymphoma (Waldenström's macroglobulinemia) cases and 30% of activated B-cell type diffuse large B-cell lymphoma (DLBCL) cases. To investigate the role of MYD88 in the growth of lymphoma/leukemia cells, with or without MYD88mutations, and to examine whether MYD88 inhibitors can be used as novel molecular-targeted drugs, we studied their effects on the growth of lymphoma and leukemia cells in vitro.

Methods : Seven lymphoma/leukemia cell lines (TMD8 derived from DLBCL with MYD88 mutations, Daudi from Burkitt lymphoma, NALM-6 from B-ALL, Jurkat and KOPT-K1 from T-ALL, THP-1 and TMD7 from AML without MYD88 mutations) and normal lymphocytes from healthy volunteers were obtained, following an informed consent. The effect of the MYD88 inhibitor, ST2825, on the in vitro growth of these cell lines was then examined using the WST-8 colorimetric assay. ST2825 is a synthetic peptide-mimetic compound, which inhibits MYD88 dimerization. A Bruton tyrosine kinase (BTK) inhibitor, ibrutinib, was used as the reference inhibitor. The effect of ST2825 on protein expression was examined by immunoblot analysis. Cell cycle analysis and Annexin V apoptosis assay were performed using flow cytometry. To comprehensively screen the changes in mRNA expression after ST2825 treatment, microarray analysis was performed for TMD8 and Jurkat cells. MYD88knockdown experiments using small interfering RNA were also performed.

Results : The MYD88 protein was expressed in all cell lines. ST2825 (30 μM) suppressed the growth of all lymphoma/leukemia cell lines, but did not affect the viability of normal lymphocytes. The IC₅₀ for TMD8 cells was similar to that for other cells, such as Daudi cells. Apoptosis assay revealed that ST2825 induces apoptosis in all the cell lines studied. In Daudi cells, ST2825 induced G2/M cell cycle arrest. Immunoblot analysis revealed that ST2825 suppresses the phosphorylation of certain NF-kB signaling components, such as RELA and IkB, in all cell lines. In TMD8, Daudi, and NALM-6 cells, ST2825 suppressed the phosphorylation of BTK, and MYD88 knockdown suppressed the phosphorylation of RELA and BTK. Microarray analysis revealed that ST2825 treatment downregulates the expression of various genes including MYD88, and upregulates the expression of other genes such as NGFR, FOSB, and HSP. Treatment with ibrutinib (1 nM) suppressed the growth of only TMD8 cells. Treatment with ST2825 plus ibrutinib additively suppressed the growth of TMD8 cells.

Conclusions : The MYD88 inhibitor ST2825 suppresses the growth of various lymphoma and leukemia cells, suggesting that MYD88 is involved in regulating the growth of these cells; however, the off-target effects of ST2825 should be considered. Further investigation is required to assess the potential of MYD88 inhibitors as novel molecular-targeted drugs against lymphoma and leukemia.