Anne M. Bowcock

Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma

Uveal melanoma is the most common primary cancer of the eye and often results in fatal metastasis. Here, we describe mutations occurring exclusively at codon 625 of the SF3B1 gene, encoding splicing factor 3B subunit 1, in low-grade uveal melanomas with good prognosis. Thus, uveal melanoma is among a small group of cancers associated with SF3B1 mutations, and these mutations denote a distinct molecular subset of uveal melanomas.

The Immunogenetics of Psoriasis: A Comprehensive Review

Psoriasis vulgaris is a common, chronic inflammatory skin disease with a complex etiology involving genetic risk factors and environmental triggers. Here we describe the many known genetic predispositions of psoriasis with respect to immune genes and their encoded pathways in psoriasis susceptibility. These genes span an array of functions that involve antigen presentation (HLA-Cw6, ERAP1, ERAP2, MICA), the IL-23 axis (IL12Bp40, IL23Ap19, IL23R, JAK2, TYK2), T-cell development and T-cells polarization (RUNX1, RUNX3, STAT3, TAGAP, IL4, IL13), innate immunity (CARD14, c-REL, TRAF3IP2, DDX58, IFIH1), and negative regulators of immune responses (TNIP1, TNFAIP3, NFKBIA, ZC3H12C, IL36RN, SOCS1). The contribution of some of these gene products to psoriatic disease has also been revealed in recent years through targeting of key immune components, such as the Th17/IL-23 axis which has been highly successful in disease treatment. However, many of the genetic findings involve immune genes with less clear roles in psoriasis pathogenesis. This is particularly the case for those genes involved in innate immunity and negative regulation of immune specific pathways. It is possible that risk alleles of these genes decrease the threshold for the initial activation of the innate immune response. This could then lead to the onslaught of the pathogenic adaptive immune response known to be active in psoriatic skin. However, precisely how these various genes affect immunobiology need to be determined and some are speculated upon in this review. These novel genetic findings also open opportunities to explore novel therapeutic targets and potentially the development of personalized medicine, as well as discover new biology of human skin disease.

Fine Mapping Major Histocompatibility Complex Associations in Psoriasis and Its Clinical Subtypes

Psoriasis vulgaris (PsV) risk is strongly associated with variation within the major histocompatibility complex (MHC) region, but its genetic architecture has yet to be fully elucidated. Here, we conducted a large-scale fine-mapping study of PsV risk in the MHC region in 9,247 PsV-affected individuals and 13,589 controls of European descent by imputing class I and II human leukocyte antigen (HLA) genes from SNP genotype data. In addition, we imputed sequence variants for MICA, an MHC HLA-like gene that has been associated with PsV, to evaluate association at that locus as well. We observed that HLA-C∗06:02 demonstrated the lowest p value for overall PsV risk (p = 1.7 × 10−364). Stepwise analysis revealed multiple HLA-C∗06:02-independent risk variants in both class I and class II HLA genes for PsV susceptibility (HLA-C∗12:03, HLA-B amino acid positions 67 and 9, HLA-A amino acid position 95, and HLA-DQα1 amino acid position 53; p < 5.0 × 10−8), but no apparent risk conferred by MICA. We further evaluated risk of two major clinical subtypes of PsV, psoriatic arthritis (PsA; n = 3,038) and cutaneous psoriasis (PsC; n = 3,098). We found that risk heterogeneity between PsA and PsC might be driven by HLA-B amino acid position 45 (pomnibus = 2.2 × 10−11), indicating that different genetic factors underlie the overall risk of PsV and the risk of specific PsV subphenotypes. Our study illustrates the value of high-resolution HLA and MICA imputation for fine mapping causal variants in the MHC.

Genome-wide Association Analysis of Psoriatic Arthritis and Cutaneous Psoriasis Reveals Differences in Their Genetic Architecture

Psoriasis vulgaris (PsV) is a common inflammatory and hyperproliferative skin disease. Up to 30% of people with PsV eventually develop psoriatic arthritis (PsA), an inflammatory musculoskeletal condition. To discern differences in genetic risk factors for PsA and cutaneous-only psoriasis (PsC), we carried out a genome-wide association study (GWAS) of 1,430 PsA case subjects and 1,417 unaffected control subjects. Meta-analysis of this study with three other GWASs and two targeted genotyping studies, encompassing a total of 9,293 PsV case subjects, 3,061 PsA case subjects, 3,110 PsC case subjects, and 13,670 unaffected control subjects of European descent, detected 10 regions associated with PsA and 11 with PsC at genome-wide (GW) significance. Several of these association signals (IFNLR1, IFIH1, NFKBIA for PsA; TNFRSF9, LCE3C/B, TRAF3IP2, IL23A, NFKBIA for PsC) have not previously achieved GW significance. After replication, we also identified a PsV-associated SNP near CDKAL1 (rs4712528, odds ratio [OR] = 1.16, p = 8.4 × 10(-11)). Among identified psoriasis risk variants, three were more strongly associated with PsC than PsA (rs12189871 near HLA-C, p = 5.0 × 10(-19); rs4908742 near TNFRSF9, p = 0.00020; rs10888503 near LCE3A, p = 0.0014), and two were more strongly associated with PsA than PsC (rs12044149 near IL23R, p = 0.00018; rs9321623 near TNFAIP3, p = 0.00022). The PsA-specific variants were independent of previously identified psoriasis variants near IL23R and TNFAIP3. We also found multiple independent susceptibility variants in the IL12B, NOS2, and IFIH1 regions. These results provide insights into the pathogenetic similarities and differences between PsC and PsA.

Driver Mutations in Uveal Melanoma: Associations With Gene Expression Profile and Patient Outcomes

IMPORTANCEnFrequent mutations have been described in the following 5 genes in uveal melanoma (UM): BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. Understanding the prognostic significance of these mutations could facilitate their use in precision medicine.nnnOBJECTIVEnTo determine the associations between driver mutations, gene expression profile (GEP) classification, clinicopathologic features, and patient outcomes in UM.nnnDESIGN, SETTING, AND PARTICIPANTSnRetrospective study of patients with UM treated by enucleation by a single ocular oncologist between November 1, 1998, and July 31, 2014.nnnMAIN OUTCOMES AND MEASURESnClinicopathologic features, patient outcomes, GEP classification (class 1 or class 2), and mutation status were recorded.nnnRESULTSnThe study cohort comprised 81 participants. Their mean age was 61.5 years, and 37% (30 of 81) were female. The GEP classification was class 1 in 35 of 81 (43%), class 2 in 42 of 81 (52%), and unknown in 4 of 81 (5%). BAP1 mutations were identified in 29 of 64 (45%), GNAQ mutations in 36 of 81 (44%), GNA11 mutations in 36 of 81 (44%), SF3B1 mutations in 19 of 81 (24%), and EIF1AX mutations in 14 of 81 (17%). Sixteen of the mutations in BAP1 and 6 of the mutations in EIF1AX were previously unreported in UM. GNAQ and GNA11 mutations were mutually exclusive. BAP1, SF3B1, and EIF1AX mutations were almost mutually exclusive with each other. Using multiple regression analysis, BAP1 mutations were associated with class 2 GEP and older patient. EIF1AX mutations were associated with class 1 GEP and the absence of ciliary body involvement. SF3B1 mutations were associated with younger patient age. GNAQ mutations were associated with the absence of ciliary body involvement and greater largest basal diameter. GNA11 mutations were not associated with any of the analyzed features. Using Cox proportional hazards modeling, class 2 GEP was the prognostic factor most strongly associated with metastasis (relative risk, 9.4; 95% CI, 3.1-28.5) and melanoma-specific mortality (relative risk, 15.7; 95% CI, 3.6-69.1) (Pu2009<u2009.001 for both). After excluding GEP class, the presence of BAP1 mutations was the factor most strongly associated with metastasis (relative risk, 10.6; 95% CI, 3.4-33.5) and melanoma-specific mortality (relative risk, 9.0; 95% CI, 2.8-29.2) (Pu2009<u2009.001 for both).nnnCONCLUSIONS AND RELEVANCEnBAP1, SF3B1, and EIF1AX mutations occur during UM tumor progression in an almost mutually exclusive manner and are associated with different levels of metastatic risk. These mutations may have value as prognostic markers in UM.

AKT inhibitors promote cell death in cervical cancer through disruption of mTOR signaling and glucose uptake

Background PI3K/AKT pathway alterations are associated with incomplete response to chemoradiation in human cervical cancer. This study was performed to test for mutations in the PI3K pathway and to evaluate the effects of AKT inhibitors on glucose uptake and cell viability. Experimental Design Mutational analysis of DNA from 140 pretreatment tumor biopsies and 8 human cervical cancer cell lines was performed. C33A cells (PIK3CAR88Q and PTENR233*) were treated with increasing concentrations of two allosteric AKT inhibitors (SC-66 and MK-2206) with or without the glucose analogue 2-deoxyglucose (2-DG). Cell viability and activation status of the AKT/mTOR pathway were determined in response to the treatment. Glucose uptake was evaluated by incubation with 18F-fluorodeoxyglucose (FDG). Cell migration was assessed by scratch assay. Results Activating PIK3CA (E545K, E542K) and inactivating PTEN (R233*) mutations were identified in human cervical cancer. SC-66 effectively inhibited AKT, mTOR and mTOR substrates in C33A cells. SC-66 inhibited glucose uptake via reduced delivery of Glut1 and Glut4 to the cell membrane. SC-66 (1 µg/ml-56%) and MK-2206 (30 µM-49%) treatment decreased cell viability through a non-apoptotic mechanism. Decreases in cell viability were enhanced when AKT inhibitors were combined with 2-DG. The scratch assay showed a substantial reduction in cell migration upon SC-66 treatment. Conclusions The mutational spectrum of the PI3K/AKT pathway in cervical cancer is complex. AKT inhibitors effectively block mTORC1/2, decrease glucose uptake, glycolysis, and decrease cell viability in vitro. These results suggest that AKT inhibitors may improve response to chemoradiation in cervical cancer.

Association of cardiovascular and metabolic disease genes with psoriasis

To the Editor: n nPsoriasis is a chronic immune-mediated and hyperproliferative disorder of the skin that affects 2–3% of the population. Psoriasis is associated with an increased incidence of several cardiovascular and metabolic co-morbidities, including coronary artery disease (CAD), hypertension, obesity, hyperlipidemia, and type 2 diabetes (T2D) (Davidovici et al., 2010). The association of these cardiovascular and metabolic diseases with psoriasis could be due to shared genetic risk variants, shared environmental triggers, activation of common inflammatory pathways, or a combination of these factors. Here, we evaluated the hypothesis that some of the increased risk of cardiovascular and metabolic diseases in psoriasis is derived from shared genetic risk factors. n nUsing the genome-wide association studies (GWAS) Catalog (available at www.genome.gov/gwastudies and accessed in December 2011), we selected 363 SNPs that showed significant association with CAD, hypertension, body mass index (BMI), hyperlipidemia (total, LDL and HDL cholesterol levels and triglyceride levels), or T2D. Selected SNPs met genome-wide significance (p < 5×10−8) in at least two GWAS or were significant in the latest meta-analysis of GWAS. Additional SNPs or loci were also included based on the latest expert opinions (McCarthy, 2010; ODonnell and Nabel, 2011; Peden and Farrall, 2011). Detailed information about the selected SNPs is provided in Table S1. n nWe evaluated the selected cardiovascular and metabolic SNPs for association with psoriasis in four psoriasis GWAS cohorts: the GAIN cohort including 1368 psoriasis cases and 1348 controls (Nair et al., 2009), an unpublished psoriasis cohort from Sweden including 725 cases and 438 controls, a Washington University/University of California San Francisco cohort including 211 psoriasis cases and 502 controls (Liu et al., 2008), and the Wellcome Trust Case-Control Consortium (WTCCC) cohort including 2178 psoriasis cases and 5175 controls (Strange et al., 2010). Further details of the Swedish cohort are described in the Supplemental Methods. IMPUTE2 was used to impute the ungenotyped SNPs by using phase 3 HapMap and 1000 Genomes pilot project CEU haplotypes as a reference. SNPTEST was used to associate the imputed dosage for each SNP with psoriasis status separately in each study population with the adjustment of the first three principal components from an MDS analysis of population stratification. The association test results for these SNPs with relatively high confidence (PROPER_Info > 0.5) were then combined by meta-analysis with the META using inverse-variance method based on a fixed-effect model. The false discovery rate method was used to correct for multiple testing (FDR_q < 0.05). n nWe first examined the associations between all selected SNPs and psoriasis status (Table 1 and Table S2). After adjusting for multiple testing with the false discovery rate method, seven SNPs were associated with psoriasis status (FDR_q < 0.05, Table 1). The alleles associated with increased risk of dyslipidemia (rs2247056, rs3177928, rs492602, and rs181362), increased blood pressure levels (rs805303, rs653178, and rs3184504), and increased CAD risk (rs3184504) were associated with increased risk of psoriasis (Table 1). The top three SNPs (rs2247056, rs3177928, and rs805303) were located in the HLA gene region which is a known psoriasis susceptibility locus. After further adjustment for the top psoriasis risk allele HLA-C*06:02 (determined by imputation as described in (Chen et al., 2012), the associations for rs2247056 and rs3177928 were mitigated (p = 0.06 and 0.07, respectively), while the association for rs805303 persisted (p = 0.005). Since multiple psoriasis risk alleles are identified in HLA loci (Chen et al., 2012), we cannot rule out the significant association for rs805303 is derived from linkage disequilibrium (LD) with other psoriasis HLA risk alleles. Interestingly, we identified 4 non-HLA SNPs with evidence of shared genetic risk between psoriasis and cardiovascular and metabolic diseases: rs492603 in FUT2, rs181362 in UBE2L3, and rs653178 and rs3184504 (in complete LD with each other) near or in SH2B3. FUT2 encodes an alpha-(1,2)fucosyltransferase that determines secretor status of blood group antigens on epithelial cells and in bodily secretions and has been recently associated with susceptibility to psoriasis and Crohns’s disease (Ellinghaus et al., 2012), type 1 diabetes (Smyth et al., 2011), primary sclerosing cholangitis (Folseraas et al., 2012), and norovirus infection (Carlsson et al., 2009). UBE2L3 encodes an ubiquitin-conjugating enzyme involved in cell proliferation and immune function and is associated with susceptibility to celiac disease and rheumatoid arthritis (Zhernakova et al., 2011), Crohn’s disease (Fransen et al., 2010), and systemic lupus erythematosus (Wang et al., 2012). The adaptor protein encoded by SH2B3 plays pleiotropic signaling roles in regulating lymphocyte differentiation, induction of VCAM-1 and E-selectin on endothelial cells by TNF-α, and thrombus formation (Devalliere and Charreau, 2011), and thus might explain its dual role in susceptibility to multiple autoimmune diseases and endothelium-related cardiovascular diseases (Jin et al., 2012). n n n nTable 1 n nTop 10 metabolic SNPs associated with psoriasis status in the meta-analysis of four studies. n n n nWith a combined 4482 psoriasis cases and 7463 controls, our meta-analysis had 80% power to detect genetic variants with OR = 1.2 at a significance level alpha of 0.05, assuming 5% of population allele frequency. Given that many cardiovascular and metabolic trait SNPs have ORs less than 1.2, we sought to determine whether cardiovascular and metabolic risk SNPs could be having a real, but more subtle effect on psoriasis susceptibility. We therefore constructed a weighted gene risk score (wGRS) to investigate the aggregate effects of the risk alleles associated with cardiovascular and metabolic traits between psoriasis patients and controls. Prior studies have shown that such genetic risk scores, which estimate an individual’s overall genetic burden, have increased ability to discriminate between cases and controls (Chen et al., 2011). The GRS were weighted according to the effect size of the risk alleles and a common set of SNPs were examined across all cohorts (see Supplemental Methods). A small but significant difference for the wGRS of CAD, total cholesterol levels, and TG levels was seen (p < 0.006 for adjusting for multiple testing of 8 traits, Table 2). The wGRS of total cholesterol levels did not reach significance after removing the top associated SNPs (rs3177928 and rs492602, p = 0.42, Table 1 and u200band2).2). No difference between psoriasis cases and controls was observed regarding the wGRS of hypertension, T2D, LDL cholesterol levels, HDL cholesterol levels, and BMI (Table 2). n n n nTable 2 n nMetabolic weighted gene risk score between controls and psoriasis cases.1 n n n nMultiple cardiovascular and metabolic co-morbidities have been observed in psoriasis patients. Here we examined whether the co-manifestation of these conditions is a result of shared genetic factors. The data presented here suggest that patients with psoriasis are enriched for certain common genetic variants (HLA, FUT2, UBE2L3, SH2B3) that predispose to increased risk of dyslipidemia, hypertension, and increased CAD risk itself. Further evaluation of these genes and pathways may be important to elucidate whether particular subsets of psoriasis patients carry a higher burden of risk for the development of these cardiovascular and metabolic co-morbidities.

Biology of advanced uveal melanoma and next steps for clinical therapeutics

Uveal melanoma is the most common intraocular malignancy although it is a rare subset of all melanomas. Uveal melanoma has distinct biology relative to cutaneous melanoma, with widely divergent patient outcomes. Patients diagnosed with a primary uveal melanoma can be stratified for risk of metastasis by cytogenetics or gene expression profiling, with approximately half of patients developing metastatic disease, predominately hepatic in location, over a 15‐yr period. Historically, no systemic therapy has been associated with a clear clinical benefit for patients with advanced disease, and median survival remains poor. Here, as a joint effort between the Melanoma Research Foundations ocular melanoma initiative, CURE OM and the National Cancer Institute, the current understanding of the molecular and immunobiology of uveal melanoma is reviewed, and on‐going laboratory research into the disease is highlighted. Finally, recent investigations relevant to clinical management via targeted and immunotherpies are reviewed, and next steps in the development of clinical therapeutics are discussed.

CARD14 Expression in Dermal Endothelial Cells in Psoriasis

Mutations in the caspase recruitment domain, family member 14 (CARD14) gene have recently been described in psoriasis patients, and explain the psoriasis susceptibility locus 2 (PSORS2). CARD14 is a scaffolding protein that regulates NF-κB activation, and psoriasis-associated CARD14 mutations lead to enhanced NF-κB signaling. CARD14 is expressed mainly in epidermal keratinocytes, but also in unidentified dermal cells. In this manuscript, the identity of the dermal cell types expressing CARD14, as well the potential functional consequence of overactive CARD14 in these dermal cell types, was determined. Using two-color immunofluorescence, dermal CARD14 did not co-localize with T-cells, dendritic cells, or macrophages. However, dermal CARD14 did highly co-localize with CD31+ endothelial cells (ECs). CARD14 was also expressed non-dermal endothelial cells, such as aortic endothelial cells, which may indicate a role of CARD14+ECs in the systemic inflammation and cardiovascular comorbidities associated with psoriasis. Additionally, phosphorylated NF-κB was found in psoriatic CARD14+ CD31+ ECs, demonstrating this pathway is active in dermal ECs in psoriasis. Transfection of dermal ECs with psoriasis-associated CARD14 mutations resulted in increased expression of several chemokines, including CXCL10, IL-8, and CCL2. These results provide preliminary evidence that CARD14 expression in ECs may contribute to psoriasis through increased expression of chemokines and facilitating recruitment of immune cells into skin.

Psoriasis mutations disrupt CARD14 autoinhibition promoting BCL10-MALT1-dependent NF-κB activation

Inherited and de novo mutations in the CARD14 gene promote the development of psoriasis, an inflammatory disease of the skin. Caspase recruitment domain-containing protein 14 (CARD14) is a member of the CARMA protein family that includes the structurally related CARD11 adaptor that mediates NF-κB activation by antigen receptors. We investigated the mechanism by which CARD14 mutation in psoriasis activates NF-κB. In contrast with wild-type CARD14, CARD14(E138A) and CARD14(G117S) psoriasis mutants interacted constitutively with BCL10 and MALT1, and triggered BCL10- and MALT1-dependent activation of NF-κB in keratinocytes. These alterations disrupted the inhibitory effect of the CARD14 linker region (LR) on NF-κB activation by facilitating BCL10 binding. Therefore, psoriasis mutations activated CARD14 by a mechanism analogous to oncogenic CARD11 mutations in non-Hodgkin B cell lymphomas. CARD14(E138A) also stimulated MALT1 paracaspase activity and activated both ERK1/2 and p38α MAP kinases. Inhibition of MALT1 with mepazine reduced CARD14(E138A)-induced expression of specific psoriasis-associated transcripts in keratinocytes. Our results establish the mechanism whereby gain-of-function CARD14 variants, which induce psoriatic disease in affected individuals, activate pro-inflammatory signalling.