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. 2014 Nov 4;111(44):E4762-8.
doi: 10.1073/pnas.1405423111. Epub 2014 Oct 23.

Interleukin 1 receptor-associated kinase 1 (IRAK1) mutation is a common, essential driver for Kaposi sarcoma herpesvirus lymphoma

Dongmei Yang  1 , Wuguo Chen  1 , Jie Xiong  2 , Carly J Sherrod  1 , David H Henry  3 , Dirk P Dittmer  4
Affiliations

Interleukin 1 receptor-associated kinase 1 (IRAK1) mutation is a common, essential driver for Kaposi sarcoma herpesvirus lymphoma

Dongmei Yang et al. Proc Natl Acad Sci U S A. .

Erratum in

Abstract

Primary effusion lymphoma (PEL) is an AIDS-defining cancer. All PELs carry Kaposi sarcoma-associated herpesvirus (KSHV). X chromosome-targeted sequencing of PEL identified 34 common missense mutations in 100% of cases. This included a Phe196Ser change in the interleukin 1 receptor-associated kinase 1 (IRAK1). The mutation was verified in primary PEL exudates. IRAK1 is the binding partner of MyD88, which is mutated in a fraction of Waldenström macroglobulinemia. Together, these two mediate toll-like receptor (TLR) signaling. IRAK1 was constitutively phosphorylated in PEL and required for survival, implicating IRAK1 and TLR signaling as a driver pathway in PEL and as a new drug development target.

Keywords: IRAK; Kaposi sarcoma; herpesviruses; myd88; primary effusion lymphoma.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Individual quality control (QC) of droplet-PCR enrichment and sequencing. Shown are six density curves representing the distribution of (A) the predicted amplicon length in the PCR, (B) the read-count per amplicon, (C) the GC-content across all PCR amplicons, (D) the mean coverage, (E) the minimal coverage, and (F) the maximal coverage on each position using the read data for the VG1 PEL as an example. (G) Coverage by GC-content. Shown are n = 8 samples and how coverage changes with amplicon GC-content. Data were from the discovery, single-lane multiplexed QC run, not the final data set, which includes additional sequencing runs. The final coverage is reported in Table 1. The vertical axis shows coverage on a log10 scale; the horizontal axis shows the %GC-content of each amplicon. Colors indicate coverage of each amplicon, with bright yellow indicative of higher and dark red of lower coverage. Note the drooping tail at >70% GC-content in all samples, which represents the maximal GC-content compatible with this type of PCR.
Fig. 2.
Fig. 2.
Heat map of transcription for highly mutated genes in PEL and other types of DLBCL. The expression values are derived from Affymetrix CEL data (GEO data set GDS1063). Red color represents high, white intermediate, and blue low expression levels. The genes shown on the vertical axis are the subset of the highly mutated genes reported in Table 2. Individual sample identifiers are shown on the bottom and classes on the top. BL, Burkitt lymphoma; CB, centroblastic; IB, immunoblastic.
Fig. 3.
Fig. 3.
IRAK1 haplotypes. In addition to the common SNV, we also uncovered multiple sample-specific SNVs across the IRAK1 coding sequence (cds). These allowed us to establish SVN haplotypes in IRAK1 for each of the samples. Shown are the positions of all SNVs across the IRAK1 targeted regions on the x axis and the sample name on the y axis. Dots indicate a nucleotide difference from the hg19 reference genome, and the color of the dots indicates the number of reads covering that specific SNV on a log10 scale. Note that all nucleotide positions were covered in all cell lines. Note that each cell line has a different SNV pattern, demonstrating the unique origin of each of our samples.
Fig. 4.
Fig. 4.
Expression of IRAK isoforms in PEL. (A) Shown is a Western blot of multiple PEL samples (BCBL-1, BC-1, BCP-1) and the BJAB Burkitt lymphoma cell line for total IRAK1, phosphoIRAK1T209, IRAK2, IRAK4, IRAK-M, and beta actin. (B) Immunofluorescence of IRAK1 phosphorylation in BJAB and BC1 cells, using antibodies against the T209 phosphorylation site in IRAK1 (green) and LANA (red). Nuclear DNA is counterstained with DAPI (blue).
Fig. 5.
Fig. 5.
Function of mutant IRAK1 in PEL. (A) BJAB and PEL cells were stimulated with 10 ng/mL IL-1β for indicated amounts of time and protein extracts were analyzed by Western blotting, using rabbit anti-IRAK1, anti-phospho IRAK1 T209, and mouse anti-β-actin, followed by secondary HRP-conjugated antibodies. (B) BJAB and PEL cells were fixed, permeabilized, and stained with rabbit anti-IRAK1 and mouse anti-LANA monoclonal antibodies, followed by fluorescence-conjugated secondary antibody. Nucleus was stained blue with DAPI. (Magnification: 600×.). (C) A panel of PEL cell lines and the BJAB Burkitt lymphoma cell line were stimulated with 10 ng/mL IL-1β for 30 min and analyzed as in A. (D) Cells viability was analyzed with a CellTiter-Glo kit. BJAB and PEL cells were infected with two independent shRNA-lentiviruses against IRAK1 for 4 d, and luminescence was measured. GFP shRNA lentiviruses were used as negative controls. (E) Result of colony formation assay at 10× and 40× magnification of BJAB and BCBL-1 cells infected with IRAK1 shRNA lentivirus vector and cultured under 5 µg/mL puromycin selection for 3 weeks. (F) Reduction in secreted IL-10 accumulated for 4 d after infection with anti-IRAK1 or anti-GFP shRNA for indicated cell lines. (G) Western blot of IRAK1 levels at 4 d after infection with anti-IRAK1 or anti-GFP shRNA for indicated cell lines.
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