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. 2011 Feb 3;470(7332):115-9.
doi: 10.1038/nature09671. Epub 2010 Dec 22.

Oncogenically active MYD88 mutations in human lymphoma

Vu N Ngo  1 , Ryan M Young, Roland Schmitz, Sameer Jhavar, Wenming Xiao, Kian-Huat Lim, Holger Kohlhammer, Weihong Xu, Yandan Yang, Hong Zhao, Arthur L Shaffer, Paul Romesser, George Wright, John Powell, Andreas Rosenwald, Hans Konrad Muller-Hermelink, German Ott, Randy D Gascoyne, Joseph M Connors, Lisa M Rimsza, Elias Campo, Elaine S Jaffe, Jan Delabie, Erlend B Smeland, Richard I Fisher, Rita M Braziel, Raymond R Tubbs, J R Cook, Denny D Weisenburger, Wing C Chan, Louis M Staudt
Affiliations

Oncogenically active MYD88 mutations in human lymphoma

Vu N Ngo et al. Nature. .

Abstract

The activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) remains the least curable form of this malignancy despite recent advances in therapy. Constitutive nuclear factor (NF)-κB and JAK kinase signalling promotes malignant cell survival in these lymphomas, but the genetic basis for this signalling is incompletely understood. Here we describe the dependence of ABC DLBCLs on MYD88, an adaptor protein that mediates toll and interleukin (IL)-1 receptor signalling, and the discovery of highly recurrent oncogenic mutations affecting MYD88 in ABC DLBCL tumours. RNA interference screening revealed that MYD88 and the associated kinases IRAK1 and IRAK4 are essential for ABC DLBCL survival. High-throughput RNA resequencing uncovered MYD88 mutations in ABC DLBCL lines. Notably, 29% of ABC DLBCL tumours harboured the same amino acid substitution, L265P, in the MYD88 Toll/IL-1 receptor (TIR) domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in other DLBCL subtypes and Burkitt's lymphoma, but was observed in 9% of mucosa-associated lymphoid tissue lymphomas. At a lower frequency, additional mutations were observed in the MYD88 TIR domain, occurring in both the ABC and germinal centre B-cell-like (GCB) DLBCL subtypes. Survival of ABC DLBCL cells bearing the L265P mutation was sustained by the mutant but not the wild-type MYD88 isoform, demonstrating that L265P is a gain-of-function driver mutation. The L265P mutant promoted cell survival by spontaneously assembling a protein complex containing IRAK1 and IRAK4, leading to IRAK4 kinase activity, IRAK1 phosphorylation, NF-κB signalling, JAK kinase activation of STAT3, and secretion of IL-6, IL-10 and interferon-β. Hence, the MYD88 signalling pathway is integral to the pathogenesis of ABC DLBCL, supporting the development of inhibitors of IRAK4 kinase and other components of this pathway for the treatment of tumours bearing oncogenic MYD88 mutations.

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Figures

Figure 1
Figure 1. MYD88 is required for survival of ABCDLBCL cells
MYD88 and IRAK1 shRNAs have selective toxicity for ABC DLBCL lines. Shown is the fraction of GFP+, shRNA-expressing cells relative to the GFP−, shRNA-negative fraction at the indicated times, normalized to the day 0 values.
Figure 2
Figure 2. MYD88 mutations in human lymphomas
a, MYD88 missense mutations in lymphoma biopsies and cell line models of ABC DLBCL (light blue), GCB DLBCL (orange), MALT lymphoma (purple) and Burkitt’s lymphoma (BL; dark blue). Amino acid positions are shown according to protein accession NP_002459. b, Frequencies of MYD88 mutations in biopsy samples from different lymphoma subtypes. c, Overlap of MYD88 mutations with other recurrent genetic alterations in ABC DLBCL tumour specimens. Genetic subsets were defined by somatic mutations and, in the case of the A20 subset, by homozygous deletion or epigenetic silencing. d, Location of MYD88 mutations in the three-dimensional structure of the MYD88 TIR domain. e, Dependence of ABC DLBCLs on MYD88 L265P. A 3′-UTR-directed MYD88 shRNA was inducibly expressed in the indicated ABC DLBCL lines, which were stably transduced with rescue vectors expressing wild-type or L265P MYD88 coding regions, or with an empty vector. Shown is the fraction of viable shRNA-expressing cells relative to the shRNA-negative fraction, normalized to day 0 values.
Figure 3
Figure 3. MYD88 mutations are gain-of-function
a, An altered IRAK1 isoform associated with MYD88 L265P. The GCB DLBCL line BJAB was transduced with GFP-tagged wild-type (WT) or L265P MYD88, or with an empty vector (null). Anti-GFP immunoprecipitates (IP) and input lysates were examined by immunoblotting for IRAK1, IRAK4 and MYD88. b, Preferential association of an altered IRAK1 isoform with MYD88 L265P. BJAB cells were transduced with the indicated GFP-tagged MYD88 isoforms and examined as in a. c,MYD88 L265P associates with phosphorylated IRAK1. Top panel: BJAB cells were transduced with the indicated GFP-tagged MYD88 isoforms. Anti-GFP immunoprecipitates were treated with λ-phosphatase as indicated and examined by immunoblotting for IRAK1 or MYD88. Bottom panel: anti-IRAK1 immunoprecipitates from HBL1 (ABC) or BJAB (GCB) cells were treated with λ-phosphatase as indicated and examined by immunoblotting for IRAK1. d, Toxicity of IRAK4 shRNAs for ABC DLBCLs. The indicated lines were transduced with retroviruses expressing IRAK4 shRNA and the relative number of shRNA+ cells is plotted versus time after shRNA induction, normalized to day 0. Data are representative of experiments with three different IRAK4 shRNAs. e, IRAK4 kinase activity is required for ABC DLBCL survival. The indicated ABC DLBCL lines were transduced with retroviruses expressing wild-type or kinase-dead IRAK4 isoforms, or with an empty vector (−ctrl). The survival of cells after induction of an IRAK4 shRNA is shown. f, A small-molecule IRAK1/4 kinase inhibitor is selectively lethal for ABC DLBCLs. Viability of the indicated lines was measured after treatment for 3 days with various inhibitor concentrations and normalized to DMSO-treated cells. g, IRAK4 kinase activity regulates IL-6 and IL-10 secretion. The indicated cytokines were measured in the supernatant of ABC DLBCL lines after treatment for 24 h with various concentrations of the IRAK1/4 inhibitor.
Figure 4
Figure 4. MYD88mutants activate NF-κB and cytokine signalling
a, Venn diagram of genes affected by MYD88 knockdown in the HBL1 ABC DLBCL line, grouped according to membership in gene expression signatures. b, MYD88 mutants constitutively activate NF-κB. BJAB cells co-expressing the indicated MYD88–GFP mutants and a NF-κB-driven luciferase reporter construct were assayed for luciferase activity, which was normalized to the expression levels of each MYD88–GFP isoform. a.u., arbitrary units. c, Correlation of MYD88 protein levels with CD83 expression. BJAB cells bearing GFP-tagged MYD88 isoforms were assessed for CD83 and GFP expression. Cells were assigned to equally sized bins based on their GFP levels, and the mean fluorescence intensity (m.f.i.) of CD83 in each bin was plotted. d, MYD88 knockdown decreases cytokine secretion in ABC DLBCL. MYD88 or control (ctrl) shRNAs were induced in ABC DLBCL lines, and the indicated cytokines were measured in the supernatant over time. e, STAT3 phosphorylation in ABC DLBCL depends on MYD88. MYD88 or control (ctrl) shRNAs were induced in ABC DLBCL lines, and cells were assessed by immunoblotting for phosphorylated STAT3 (pY-STAT3), total STAT3, MYD88 and β-actin. f, Preferential association of MYD88 mutant isoforms with the STAT3-high subgroup of ABC DLBCL tumours. See text for details. g, MYD88 and B-cell-receptor signalling pathways cooperate to maintain ABC DLBCL survival. OCI-Ly10 ABC DLBCL cells were first transduced with retroviruses expressing MYD88 or control shRNAs and then infected with retroviruses expressing CD79A, CARD11, or control shRNAs along with GFP. The relative viability of GFP+ cells is plotted, normalized to day 0 values. All error bars are s.e.m. (n = 3).
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