Essential genes shape cancer genomes through linear limitation of homozygous deletions.

The landscape of somatic acquired deletions in cancer cells is shaped by positive and negative selection. Recurrent deletions typically target tumor suppressor, leading to positive selection. Simultaneously, loss of a nearby essential gene can lead to negative selection, and introduce latent vulnerabilities specific to cancer cells. Here we show that, under basic assumptions on positive and negative selection, deletion limitation gives rise to a statistical pattern where the frequency of homozygous deletions decreases approximately linearly between the deletion target gene and the nearest essential genes. Using DNA copy number data from 9,744 human cancer specimens, we demonstrate that linear deletion limitation exists and exposes deletion-limiting genes for seven known deletion targets (CDKN2A, RB1, PTEN, MAP2K4, NF1, SMAD4, and LINC00290). Downstream analysis of pooled CRISPR/Cas9 data provide further evidence of essentiality. Our results provide further insight into how the deletion landscape is shaped and identify potentially targetable vulnerabilities.

The multiple myeloma risk allele at 5q15 lowers ELL2 expression and increases ribosomal gene expression.

Recently, we identified ELL2 as a susceptibility gene for multiple myeloma (MM). To understand its mechanism of action, we performed expression quantitative trait locus analysis in CD138+ plasma cells from 1630 MM patients from four populations. We show that the MM risk allele lowers ELL2 expression in these cells (Pcombined = 2.5 × 10-27; ßcombined = -0.24 SD), but not in peripheral blood or other tissues. Consistent with this, several variants representing the MM risk allele map to regulatory genomic regions, and three yield reduced transcriptional activity in plasmocytoma cell lines. One of these (rs3777189-C) co-locates with the best-supported lead variants for ELL2 expression and MM risk, and reduces binding of MAFF/G/K family transcription factors. Moreover, further analysis reveals that the MM risk allele associates with upregulation of gene sets related to ribosome biogenesis, and knockout/knockdown and rescue experiments in plasmocytoma cell lines support a cause-effect relationship. Our results provide mechanistic insight into MM predisposition.

Identification of sequence variants influencing immunoglobulin levels.

Immunoglobulins are the effector molecules of the adaptive humoral immune system. In a genome-wide association study of 19,219 individuals, we found 38 new variants and replicated 5 known variants associating with IgA, IgG or IgM levels or with composite immunoglobulin traits, accounted for by 32 loci. Variants at these loci also affect the risk of autoimmune diseases and blood malignancies and influence blood cell development. Notable associations include a rare variant at RUNX3 decreasing IgA levels by shifting isoform proportions (rs188468174[C>T]: P = 8.3 × 10-55, ß = -0.90 s.d.), a rare in-frame deletion in FCGR2B abolishing IgG binding to the encoded receptor (p.Asn106del: P = 4.2 × 10-8, ß = 1.03 s.d.), four IGH locus variants influencing class switching, and ten new associations with the HLA region. Our results provide new insight into the regulation of humoral immunity.

Direct evidence for a polygenic etiology in familial multiple myeloma.

Although common risk alleles for multiple myeloma (MM) were recently identified, their contribution to familial MM is unknown. Analyzing 38 familial cases identified primarily by linking Swedish nationwide registries, we demonstrate an enrichment of common MM risk alleles in familial compared with 1530 sporadic cases (P = 4.8 × 10-2 and 6.0 × 10-2, respectively, for 2 different polygenic risk scores) and 10 171 population-based controls (P = 1.5 × 10-4 and 1.3 × 10-4, respectively). Using mixture modeling, we estimate that about one-third of familial cases result from such enrichments. Our results provide the first direct evidence for a polygenic etiology in a familial hematologic malignancy.

Genome-wide association study identifies multiple susceptibility loci for multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy with a significant heritable basis. Genome-wide association studies have transformed our understanding of MM predisposition, but individual studies have had limited power to discover risk loci. Here we perform a meta-analysis of these GWAS, add a new GWAS and perform replication analyses resulting in 9,866 cases and 239,188 controls. We confirm all nine known risk loci and discover eight new loci at 6p22.3 (rs34229995, P=1.31 × 10(-8)), 6q21 (rs9372120, P=9.09 × 10(-15)), 7q36.1 (rs7781265, P=9.71 × 10(-9)), 8q24.21 (rs1948915, P=4.20 × 10(-11)), 9p21.3 (rs2811710, P=1.72 × 10(-13)), 10p12.1 (rs2790457, P=1.77 × 10(-8)), 16q23.1 (rs7193541, P=5.00 × 10(-12)) and 20q13.13 (rs6066835, P=1.36 × 10(-13)), which localize in or near to JARID2, ATG5, SMARCD3, CCAT1, CDKN2A, WAC, RFWD3 and PREX1. These findings provide additional support for a polygenic model of MM and insight into the biological basis of tumour development.

Increased risk of acute myocardial infarction and elevated levels of C-reactive protein in carriers of the Thr-87 variant of the ATP receptor P2Y11.

AIMS: Extracellular ATP acting on the P2Y11 receptor regulates inflammatory cells. We hypothesized that polymorphisms in the receptor could influence the risk of acute myocardial infarction (AMI). METHODS AND RESULTS: In the Malmö diet and cancer AMI case-control study (n = 3732) the P2Y11 gene Thr-87 polymorphism was present in 19.8% of the controls and 22.9% in AMI patients (OR 1.21; P = 0.03). Stronger associations were found in patients with family history (FH) of AMI, 1.32; early-onset (EO) AMI, 1.43; or EO AMI combined with FH, 1.50; supporting a genetic mechanism. The Thr-87 homozygotes had an even greater risk of AMI, 1.94 (P = 0.04); and 2.48 in the EO AMI subgroup, suggesting a genetic dosage effect. In the cardiovascular risk factor group (n = 6055), 21.3% carried the Thr-87 allele. C-reactive protein was elevated in Thr-87 carriers: 1.6 mg/L vs. 1.3 mg/L (P = 0.001). No difference was seen for blood pressure, lipids, body mass index, smoking, or diabetes mellitus. CONCLUSION: The common Ala-87-Thr polymorphism of the P2Y11 receptor is associated with AMI and increased levels of C-reactive protein. We hypothesize that an inflammatory mechanism might be involved. The P2Y11 receptor is a promising new drug target in the prevention of AMI.

Residual platelet ADP reactivity after clopidogrel treatment is dependent on activation of both the unblocked P2Y(1) and the P2Y (12) receptor and is correlated with protein expression of P2Y (12).

Two ADP receptors have been identified on human platelets: P2Y(1) and P2Y(12). The P2Y(12) receptor blocker clopidogrel is widely used to reduce the risks in acute coronary syndromes, but, currently, there is no P2Y(1) blocker in clinical use. Evidence for variable responses to clopidogrel has been described in several reports. The mechanistic explanation for this phenomenon is not fully understood. The aim of this study was to examine mechanisms responsible for variability of 2MeS-ADP, a stable ADP analogue, induced platelet reactivity in clopidogrel-treated patients. Platelet reactivity was assessed by flow cytometry measurements of P-selectin (CD62P) and activated GpIIb/IIIa complex (PAC-1). Residual 2MeS-ADP activation via the P2Y(12) and P2Y(1) receptors was determined by co-incubation with the selective antagonists AR-C69931 and MRS2179 in vitro. P2Y(1) and P2Y(12) receptor expression on both RNA and protein level were determined, as well as the P2Y(12) H1 or H2 haplotypes. Our data suggest that the residual platelet activation of 2MeS-ADP after clopidogrel treatment is partly due to an inadequate antagonistic effect of clopidogrel on the P2Y(12) receptor and partly due to activation of the P2Y(1) receptor, which is unaffected by clopidogrel. Moreover, a correlation between increased P2Y(12) protein expression on platelets and decreased response to clopidogrel was noticed, r(2)=0.43 (P<0.05). No correlation was found between P2Y(12) mRNA levels and clopidogrel resistance, indicating post-transcriptional mechanisms. To achieve additional ADP inhibition in platelets, antagonists directed at the P2Y(1) receptor could be more promising than the development of more potent P2Y(12) receptor antagonists.

Positive inotropic effects by uridine triphosphate (UTP) and uridine diphosphate (UDP) via P2Y2 and P2Y6 receptors on cardiomyocytes and release of UTP in man during myocardial infarction.

The aim of this study was to examine a possible role for extracellular pyrimidines as inotropic factors for the heart. First, nucleotide plasma levels were measured to evaluate whether UTP is released in patients with coronary heart disease. Then, inotropic effects of pyrimidines were examined in isolated mouse cardiomyocytes. Finally, expression of pyrimidine-selective receptors (a subgroup of the P2 receptors) was studied in human and mouse heart, using real time polymerase chain reaction, Western blot, and immunohistochemistry. Venous plasma levels of UTP were increased (57%) in patients with myocardial infarction. In electrically stimulated cardiomyocytes the stable P2Y(2/4) agonist UTPgammaS increased contraction by 52%, similar to beta1-adrenergic stimulation with isoproterenol (65%). The P2Y6-agonist UDPbetaS also increased cardiomyocyte contraction (35%), an effect abolished by the P2Y6-blocker MRS2578. The phospholipase C inhibitor U73122 inhibited both the UDPbetaS and the UTPgammaS-induced inotropic effect, indicating an IP3-mediated effect via P2Y6 receptors. The P2Y14 agonist UDP-glucose was without effect. Quantification of mRNA with real time polymerase chain reaction revealed P2Y2 as the most abundant pyrimidine receptor expressed in cardiomyocytes from man. Presence of P2Y6 receptor mRNA was detected in both species and confirmed at protein level with Western blot and immunohistochemistry in man. In conclusion, UTP levels are increased in humans during myocardial infarction, giving the first evidence for UTP release in man. UTP is a cardiac inotropic factor most likely by activation of P2Y2 receptors in man. For the first time we demonstrate inotropic effects of UDP, mediated by P2Y6 receptors via an IP3-dependent pathway. Thus, the extracellular pyrimidines (UTP and UDP) could be important inotropic factors involved in the development of cardiac disease.

Phospholipase C and cAMP-dependent positive inotropic effects of ATP in mouse cardiomyocytes via P2Y11-like receptors.

ATP is released as a cotransmitter together with catecholamines from sympathetic nerves. In the heart ATP has been shown to cause a pronounced positive inotropic effect and may also act in synergy with beta-adrenergic agonists to augment cardiomyocyte contractility. The aim of the present study was to investigate the inotropic effects mediated by purinergic P2 receptors using isolated mouse cardiomyocytes. Stable adenine nucleotide analogs were used and the agonist rank order for adenine nucleotide stimulation of the mouse cardiomyocytes was AR-C67085>ATPgammaS>2-MeSATP>>>2-MeSADP=0, that fits the agonist profile of the P2Y11 receptor. ATPgammaS induced a positive inotropic response in single mouse cardiomyocytes. The response was similar to that for the beta1 receptor agonist isoproterenol. The most potent response was obtained using AR-C67085, a P2Y11 receptor agonist. This agonist also potentiated contractions in isolated trabecular preparations. The adenylyl cyclase blocker (SQ22563) and phospholipase C (PLC) blocker (U73122) demonstrated that both pathways were required for the inotropic response of AR-C67085. A cAMP enzyme immunoassay confirmed that AR-C67085 increased cAMP in the cardiomyocytes. These findings are in agreement with the P2Y11 receptor, coupled both to activation of IP3 and cAMP, being a major receptor for ATP induced inotropy. Analyzing cardiomyocytes from desmin deficient mice, Des-/-, with a congenital cardiomyopathy, we found a lower sensitivity to AR-C67085, suggesting a down-regulation of P2Y11 receptor function in heart failure. The prominent action of the P2Y11 receptor in controling cardiomyocyte contractility and possible alterations in its function during cardiomyopathy may suggest this receptor as a potential therapeutic target. It is possible that agonists for the P2Y11 receptor could be used to improve cardiac output in patients with circulatory shock and that P2Y11 receptor antagonist could be beneficial in patients with congestive heart failure (CHF).

Plasticity of TRPC expression in arterial smooth muscle: correlation with store-operated Ca2+ entry.

Loss of the smooth muscle contractile phenotype is critical in atherosclerosis and in restenosis after angioplasty, but its early signals are incompletely understood. In this study, we have explored the role of transient receptor potential canonical (TRPC) proteins, which have been suggested to mediate store-operated Ca2+ entry (SOCE). Contractility of rat cerebral arteries in organ culture is preserved for several days, whereas SOCE is increased. In correlation with this increase is that nifedipine-insensitive whole cell current, activated by depletion of intracellular Ca2+ stores, was increased by 50% in cells isolated from arteries cultured for 3 days. TRPC1 and TRPC6 mRNA were more than fivefold increased in cells isolated after organ culture, whereas TRPC3 was decreased. Immunofluorescent staining and/or Western blotting of arteries and isolated cells showed upregulation of TRPC1 and TRPC6 proteins during organ culture. In intact arteries, TRPC4 expression correlated with the amount of endothelium present. Ca2+ addition after store depletion caused a contraction in cultured, but not in freshly dissected, arteries. A polyclonal TRPC1 antibody directed against an extracellular epitope inhibited this contraction by approximately 50%. To investigate the basis of the TRPC upregulation and assess its possible clinical significance, segments of human internal mammary artery were organ cultured for 24 h and then exposed to balloon dilatation in vitro, followed by further culturing for up to 48 h. After dilatation, TRPC1 and TRPC6 mRNA were progressively increased compared with undilated control segments. The results of this study indicate that vascular injury enhances plasticity in TRPC expression, that TRPC expression correlates with cellular Ca2+ handling, and that TRPC1 is a subunit of upregulated store-operated Ca2+ channels.

ADP receptor P2Y12 is expressed in vascular smooth muscle cells and stimulates contraction in human blood vessels.

OBJECTIVE: ADP plays an important role in platelet aggregation by activating P2Y12 receptors. We assessed the hypothesis that P2Y12 receptors are expressed in vascular smooth muscle cells (VSMC). METHODS AND RESULTS: P2Y12 receptor mRNA was found to have a high expression among the P2 receptors in human VSMC, significantly higher than the other 2 ADP receptors (P2Y1 and P2Y13, real-time polymerase chain reaction). Western blots gave a band of 50 kD, similar to that in platelets. To unmask a P2Y12 receptor-mediated vasoconstriction by simulating the in vivo situation, vessels were precontracted to a submaximal level. 2-MeSADP stimulated contractions in vessel segments from internal mammary artery (IM), IM branches and small veins (Emax=15+/-6% of 60 mmol/L K+ contraction, pEC50=5.6+/-0.6, Emax=21+/-1%, pEC50=6.8+/-0.1, and Emax=48+/-9%, pEC50=6.6+/-0.4). The selective P2Y12 antagonist AR-C67085 blocked 2-MeSADP contractions. The contraction was not reduced in patients using clopidogrel, a drug inhibiting ADP-induced platelet aggregation by blocking the P2Y12 receptor. This may be explained by the high instability of the active clopidogrel metabolite that never reaches the systemic circulation. CONCLUSIONS: ADP acting on P2Y12 receptors not only is important for platelet activation but also stimulates vasoconstriction. Stable drugs with antagonistic effects on P2Y12 receptors, affecting both platelets and VSMC, could be of double therapeutic benefit in their prevention of both thrombosis and vasospasm.

Extracellular nucleotides induce vasodilatation in human arteries via prostaglandins, nitric oxide and endothelium-derived hyperpolarising factor.

1. The present study was aimed at examining P2 receptor-mediated vasodilatation in human vessels. The isometric tension was recorded in isolated segments of the human left internal mammary artery branches precontracted with 1 microM noradrenaline. 2. Endothelial denudation abolished the dilator responses. 3. The selective P2Y(1) agonist, 2-MeSADP, induced a potent vasodilatation (pEC(50)=6.9+/-0.1). The P2Y(1) antagonist of 10 microM, MRS 2216, shifted the 2-MeSADP concentration-response curve 1.1 log units to the right. The combined P2Y(1) and P2X agonist, 2-MeSATP, stimulated a dilatation with a potency similar to that of 2-MeSADP. Furthermore, MRS 2216 had a similar antagonistic effect on both 2-MeSATP and 2-MeSADP indicating that P2X receptors do not mediate vasodilatation. 4. Both the P2Y(2/4) agonist, UTPgammaS and the P2Y(6) agonist, UDPbetaS, stimulated potent dilatations (pEC(50)=7.8+/-0.4 for UTPgammaS and 8.4+/-0.2 for UDPbetaS). 5. The 2-MeSADP-induced nitric oxide (NO)-mediated dilatation was studied in the presence of 10 micro M indomethacin, 50 nM charybdotoxin and 1 microM apamin. The involvement of the endothelium-derived hyperpolarising factor (EDHF) was investigated in the presence of 0.1 mM L-NOARG and indomethacin. The involvement of prostaglandins was investigated in the presence of L-NOARG, charybdotoxin and apamin. Both NO, EDHF and prostaglandins mediated 2-MeSADP dilatation with similar efficacy (E(max)=25+/-5% for NO, 25+/-6% for EDHF and 27+/-5% for prostaglandins). 6. In conclusion, extracellular nucleotides induce endothelium-derived vasodilatation in human vessels by stimulating P2Y(1), P2Y(2/4) and P2Y(6) receptors, while P2X receptors are not involved. Endothelial P2Y receptors mediate dilatation by release of EDHF, NO and prostaglandins.

Methanocarba modification of uracil and adenine nucleotides: high potency of Northern ring conformation at P2Y1, P2Y2, P2Y4, and P2Y11 but not P2Y6 receptors.

The potency of nucleotide antagonists at P2Y1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y1 or human P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y4 receptors with an EC(50) of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y4 response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y1, P2Y2, P2Y4, and P2Y11 receptors.