Project description:The ABC subtype of diffuse large B cell lymphoma (DLBCL) remains the least curable form of this lymphoma despite recent advances in therapy. We have combined structural and functional genomics to triangulate on new oncogenic mechanisms and devise new therapeutic strategies. RNA interference screen revealed a dependence of ABC DLBCL cell lines on MYD88 and IRAK1. High throughput resequencing of RNA (RNA-Seq) revealed frequent somatic mutations in MYD88 that preferentially occurred in the ABC DLBCL subtype. Remarkably, one third of ABC DLBCL tumor samples harbored the same amino acid substitution, L265P, in the MYD88 TIR domain at an evolutionarily invariant residue in its hydrophobic core. This mutation was rare or absent in two other DLBCL subtypes, but was observed in 9% of MALT lymphomas. At a lower frequency, multiple other mutations were observed in the MYD88 TIR domain, occurring in both the ABC and GCB subtypes of DLBCL. Survival of ABC DLBCL lines bearing the L265P mutation was sustained by the mutant but not wild type MYD88 isoform, demonstrating that this MYD88 mutant is oncogenic and gain-of-function. The MYD88 L265P mutant assembled a protein complex that spontaneous triggers the phosphorylation of IRAK1, leading to NF-kB signaling, secretion of the cytokines IL-6, IL-10 and interferon-b, and JAK kinase signaling. These findings demonstrate that the MYD88 signaling pathway is integral to the pathogenesis of ABC DLBCL, providing a genetic rationale for therapeutic targeting of the MYD88 signaling pathway in this lymphoma subtype. To generate a gene expression signature of MYD88 signaling in ABC DLBCL, the HBL-1 cell line was transduced with retroviral vectors expressing either shMYD88-4 or shMYD88-7. Following puromycin selection, shRNA expression was induced for 24 or 48 hours and gene expression was measured, comparing uninduced (Cy3) to induced (Cy5) cells, using genome-wide Agilent 4x44K oligonucleotide microarrays. A signature of NF-kB signaling in ABC DLBCL was generated by treating HBL-1 cells with the IkB kinase beta inhibitor MLN120B for 2h, 3h, 4h, 6h, 8h, 12h, 16h, and 24h (Cy5), and comparing their gene expression to untreated cells (Cy3). A signature of JAK signaling in ABC DLBCL was generated by treating HBL-1 cells with JAK inhibitor I (5 micromolar; Calbiochem) for 2h, 4h, 6h, and 8h (Cy5) and comparing their gene expression to vehicle-treated cells (DMSO, Cy3). RNA-Seq data not provided.

Project description:Next-generation sequencing has revolutionized cancer biology by accelerating the unbiased discovery of novel mutations across human cancers. Transforming such discoveries into a conceptual framework of cancer progression requires narrowing the vast number of mutations down to the driver elements, and further reducing these to mutations that govern cancer progression as opposed to tumor initiation. By integrating next-generation RNA-sequencing (RNA-seq) with in vivo selection, we devise an approach that identifies a series of novel recurrent non-synonymous amino acid mutations that are enriched in metastatic breast cancer cells and predicted to significantly alter protein function. These mutations, found in PANX1, RBFA, REST, KRIT1 and ZSWIM6, are detected at higher frequencies in the transcriptomes of two patientsâ highly metastatic sub-lines relative to their poorly metastatic parental lines. We functionally characterize the cellular and molecular roles of one of these mutationsâa nonsense alteration that yields a truncated pannexin-1 (PANX11-89) plasma membrane megachannel subunitâin metastatic progression. PANX11-89 interacts with full-length PANX1 and augments PANX1 channel activity to promote the survival of cancer cells as they are mechanically deformed. Protection from deformation-induced cell death requires PANX1 channels to release ATP, which acts as a cell autonomous survival signal during mechanical stress. Functional characterization of additional nonsense and missense PANX1 mutations detected in epithelial cancers of the colon, lung, and prostate reveals that these mutants also enhance PANX1-mediated ATP release. In vivo testing of one such truncating mutation detected in a metastatic colorectal tumor also enhances early survival, dissemination and liver metastatic colonization by human colon cancer cells. Finally, pharmacological inhibition of PANX1 inhibits breast cancer metastasis, implicating PANX1 as a novel therapeutic target in cancer. Our findings reveal that ATP release through mechanosensitive PANX1 channels enables cancer cells to overcome a major metastasis suppressive barrier—deformation-induced death in the microvasculature. To systematically identify base-pair mutations present in metastatic cells that may drive cancer progression, we performed whole-transcriptome RNA-sequencing (RNA-seq) of in vivo-selected, highly metastatic human breast cancer cell sub-lines, CN-LM1A and MDA-LM2, as well as the CN34 and MDA-MB-231 parental lines from which they were derived. To minimize the false positive rate and allow for subsequent statistical analysis, we sequenced biological replicates of each cell line. Mutations conferring enhanced metastatic capacity should be enriched in the transcriptomes of highly metastatic cells relative to their less metastatic parental populations.

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Homo sapiens

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