Ubiquitin-like protein 3 (UBL3) is required for MARCH ubiquitination of major histocompatibility complex class II and CD86.

The MARCH E3 ubiquitin (Ub) ligase MARCH1 regulates trafficking of major histocompatibility complex class II (MHC II) and CD86, molecules of critical importance to immunity. Here we show, using a genome-wide CRISPR knockout screen, that ubiquitin-like protein 3 (UBL3) is a necessary component of ubiquitination-mediated trafficking of these molecules in mice and in humans. Ubl3-deficient mice have elevated MHC II and CD86 expression on the surface of professional and atypical antigen presenting cells. UBL3 also regulates MHC II and CD86 in human dendritic cells (DCs) and macrophages. UBL3 impacts ubiquitination of MARCH1 substrates, a mechanism that requires UBL3 plasma membrane anchoring via prenylation. Loss of UBL3 alters adaptive immunity with impaired development of thymic regulatory T cells, loss of conventional type 1 DCs, increased number of trogocytic marginal zone B cells, and defective in vivo MHC II and MHC I antigen presentation. In summary, we identify UBL3 as a conserved, critical factor in MARCH1-mediated ubiquitination with important roles in immune responses.

Somatic mutation footprinting reveals a unique tetranucleotide signature associated with intron-exon boundaries in lung cancer.

Cigarette smoke comprises a large number of carcinogenic substances that can increase DNA mutation load in epithelial cells of the mouth, throat and lungs. While a strong C:A substitution preference is abundant in tobacco-related cancer genomes, detection of complex or less abundant somatic mutation signatures may be confounded by the heterogeneity of carcinogens present in smoke. Trinucleotide signatures are defined for a variety of somatic mutation processes, yet the extent to which this configuration optimally defines and discriminates between mutational processes is not clear. Here, we describe a method that determines whether trinucleotide patterns do a good job at encapsulating a mutation signature or whether they mask underlying heterogeneity that alternative pattern structures would better define. The approach works by mapping the dependency of trinucleotide signatures in relation to sequence context to establish a 'footprint' of context dependency. Applying this technique to smoke-associated cancers, we show that a robust tetranucleotide substitution is prevalent in 17% of lung squamous cell carcinoma genomes. The signature is dominated by the substitution CT(C:A)G and is strongly associated with gene expression level and intron-exon junctions. Intriguingly, its distribution across the genome is biased towards 5' splice junctions, suggesting a novel mechanism of mutation.

Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer.

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFß, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses.

Association of candidate single nucleotide polymorphisms with somatic mutation of the epidermal growth factor receptor pathway.

BACKGROUND: Tumour growth in colorectal cancer and other solid cancers is frequently supported by activating mutations in the epidermal growth factor receptor (EGFR) signaling pathway (Patholog Res Int 2011:932932, 2011). Treatment of metastatic colorectal cancer with targeted anti-EGFR therapeutics such as cetuximab extends survival in only 25% of patients who test wild-type for KRAS, while the majority of patients prove resistant (J Clin Oncol 28(7):1254-1261, 2010).Prediction of cetuximab responsiveness for KRAS wild-type colorectal cancers is currently not well defined, and prognostic biomarkers would help tailor treatment to individual patients. Somatic mutation of the EGFR signalling pathway is a prevalent mechanism of resistance to cetuximab (Nature 486(7404):532-536, 2012). If the human genome harbours variants that influence susceptibility of the EGFR pathway to oncogenic mutation, such variants could also be prognostic for cetuximab responsiveness. METHODS: We assessed whether patient genetic variants may associate with somatic mutation of the EGFR signalling pathway. We combined tumour mutation data from the Cancer Genome Atlas with matched patient genetic data, and tested for germline variants that associate with somatic mutation of the EGFR pathway (including EGFR, KRAS, BRAF, PTEN and PIK3CA). RESULTS: Two single nucleotide polymorphisms (SNPs) located 90 kb upstream of the TERT oncogene associated with somatic mutation of the EGFR pathway beyond the threshold of genome-wide significance: rs7736074 (P = 4.64 × 10-9) and rs4975596 (P = 5.69 × 10-9). We show that allelic variants of rs7736074 and rs4975596 modulate TERT expression levels in multiple cancer types, and exhibit preliminary prognostic value for response to cetuximab. CONCLUSIONS: We have identified two germline SNPs that associate with somatic mutation of the EGFR pathway, and may be prognostic for cetuximab responsiveness. These variants could potentially contribute to a panel of prognostic biomarkers for assessing whether metastatic colorectal cancer patients are likely to derive benefit from cetuximab treatment. Genotyping of a large cohort of cetuximab-treated colorectal cancer patients is called for to further clarify the association.

The negative regulatory roles of suppressor of cytokine signaling proteins in myeloid signaling pathways.

PURPOSE OF REVIEW: Suppressor of cytokine signaling proteins are key regulators of the response of myeloid cells to cytokines and other extracellular stimuli. This review explores recent developments that have shed light on how two of the best-characterized suppressor of cytokine signaling proteins, SOCS1 and SOCS3, attenuate myeloid signaling pathways that lead to inflammation, hematological malignancy, and related disorders. RECENT FINDINGS: In addition to its 'classic' role as an inhibitor of IFNgamma signaling, a number of new regulatory roles in IFNalpha and toll-like receptor signaling have been defined for SOCS1, and substantial progress has been made in identifying the factors that give rise to lethal inflammation in Socs1 neonatal mice. The aberrant transcriptional regulation of suppressor of cytokine signaling genes in myeloid leukemia and related proliferative disorders has also been further defined. Finally, positive signs have emerged in mice that exogenous delivery of SOCS3 may be of therapeutic value. SUMMARY: Suppressor of cytokine signaling proteins have pivotal roles in attenuating cytokine and toll-like receptor signaling in myeloid cells. Understanding how defective suppressor of cytokine signaling activity contributes to inflammatory and malignant disease promises to create significant new therapeutic opportunities.

Proximal genomic localization of STAT1 binding and regulated transcriptional activity.

BACKGROUND: Signal transducer and activator of transcription (STAT) proteins are key regulators of gene expression in response to the interferon (IFN) family of anti-viral and anti-microbial cytokines. We have examined the genomic relationship between STAT1 binding and regulated transcription using multiple tiling microarray and chromatin immunoprecipitation microarray (ChIP-chip) experiments from public repositories. RESULTS: In response to IFN-gamma, STAT1 bound proximally to regions of the genome that exhibit regulated transcriptional activity. This finding was consistent between different tiling microarray platforms, and between different measures of transcriptional activity, including differential binding of RNA polymerase II, and differential mRNA transcription. Re-analysis of tiling microarray data from a recent study of IFN-gamma-induced STAT1 ChIP-chip and mRNA expression revealed that STAT1 binding is tightly associated with localized mRNA transcription in response to IFN-gamma. Close relationships were also apparent between STAT1 binding, STAT2 binding, and mRNA transcription in response to IFN-alpha. Furthermore, we found that sites of STAT1 binding within the Encyclopedia of DNA Elements (ENCODE) region are precisely correlated with sites of either enhanced or diminished binding by the RNA polymerase II complex. CONCLUSION: Together, our results indicate that STAT1 binds proximally to regions of the genome that exhibit regulated transcriptional activity. This finding establishes a generalized basis for the positioning of STAT1 binding sites within the genome, and supports a role for STAT1 in the direct recruitment of the RNA polymerase II complex to the promoters of IFN-gamma-responsive genes.

The comparative roles of suppressor of cytokine signaling-1 and -3 in the inhibition and desensitization of cytokine signaling.

Negative feedback is a mechanism commonly employed in biological processes as a means of maintaining homeostasis. We have investigated the roles of suppressor of cytokine signaling (SOCS) proteins in regulating the kinetics of negative feedback in response to cytokine signaling. In mouse livers and bone marrow-derived macrophages, both interferon-gamma (IFNgamma) and interleukin-6 (IL-6) rapidly induced the tyrosine phosphorylation of signal transducer and activator of transcription-1 (STAT1) and STAT3. STAT3 tyrosine phosphorylation was bi-phasic in response to continuous IL-6 signaling. In macrophages lacking Socs3, however, continuous IL-6 signaling induced uniformly high levels of STAT3 tyrosine phosphorylation, and early IL-6-inducible genes were inappropriately expressed at intermediate time points. SOCS3 therefore imposes bi-phasic kinetics upon IL-6 signaling. Compared with Socs3 mRNA, Socs1 mRNA was induced relatively slowly, and SOCS1 simply attenuated the duration of IFNgamma signaling. Surprisingly, heightened Socs1 mRNA expression but minimal STAT1 tyrosine phosphorylation was observed after prolonged stimulation with IFNgamma, indicating that STAT1 may not play a large role in inducing Socs1 mRNA during steady-state IFNgamma signaling. We also demonstrate that both SOCS1 and SOCS3 can desensitize primary bone marrow-derived macrophages to IFNgamma and IL-6 signaling, respectively. Consistent with the kinetics with which Socs1 and Socs3 mRNAs were induced, SOCS3 desensitized cells to IL-6 rapidly, whereas SOCS1-mediated desensitization to IFNgamma occurred at later time points. The kinetics with which SOCS proteins are induced by cytokine may therefore be a parameter that is "hard-wired" into specific cytokine signaling pathways as a means of tailoring the kinetics with which cells become desensitized.

Inhibitors of cytokine signal transduction.

Cytokines are secreted proteins that regulate diverse biological functions by binding to receptors at the cell surface to activate complex signal transduction pathways including the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Stringent mechanisms of signal attenuation are essential for ensuring an appropriate, controlled cellular response. Three families of proteins, the SH2-containing phosphatases (SHP), the protein inhibitors of activated STATs (PIAS), and the suppressors of cytokine signaling (SOCS), inhibit specific and distinct aspects of cytokine signal transduction. The analysis of mice lacking genes for members of the SHP and SOCS families has shed much light on the roles of these proteins in vivo. In recent in vitro studies, the protein modifiers ubiquitin and SUMO (small ubiquitin-like modifier) have emerged as key players in the strategies employed by SOCS and PIAS to repress signaling.