Activation Fusion Team 2005 Activation EXCLUSIVE

Aneutronic fusion is any form of fusion power in which very little of the energy released is carried by neutrons. While the lowest-threshold nuclear fusion reactions release up to 80% of their energy in the form of neutrons, aneutronic reactions release energy in the form of charged particles, typically protons or alpha particles. Successful aneutronic fusion would greatly reduce problems associated with neutron radiation such as damaging ionizing radiation, neutron activation, reactor maintenance, and requirements for biological shielding, remote handling and safety.

A French research team fused protons and boron-11 nuclei using a laser-accelerated proton beam and high-intensity laser pulse.[13] In October 2013 they reported an estimated 80 million fusion reactions during a 1.5 nanosecond laser pulse.[13]

Myeloproliferative disorders (MPDs) are heterogeneous diseases that occur at the level of a multipotent hematopoietic stem cell. They are characterized by increased blood cell production related to cytokine hypersensitivity and virtually normal cell maturation. The molecular pathogenesis of the MPDs has been poorly understood, except for chronic myeloid leukemia (CML), where the Bcr-Abl fusion protein exhibits constitutive kinase activity. Since some rare MPDs are also related to a dysregulated kinase activity, a similar mechanism was thought to be likely responsible for the more frequent MPDs. We investigated the mechanisms of endogenous erythroid colony formation (EEC) by polycythemia vera (PV) erythroid progenitor cells and found that EEC formation was abolished by a pharmacological inhibitor of JAK2 as well as an siRNA against JAK2. JAK2 sequencing revealed a unique mutation in the JH2 domain leading to a V617F substitution in more than 80% of the PV samples. This mutation in the pseudokinase autoinhibitory domain results in constitutive kinase activity and induces cytokine hypersensitivity or independence of factor-dependent cell lines. Retroviral transduction of the mutant JAK2 into murine HSC leads to the development of an MPD with polycythemia. The same mutation was found in about 50% of patients with idiopathic myelofibrosis (IMF) and 30% of patients with essential thrombocythemia (ET). Using different approaches, four other teams have obtained similar results. The identification of the JAK2 mutation represents a major advance in our understanding of the molecular pathogenesis of MPDs that will likely permit a new classification of these diseases and the development of novel therapeutic approaches.

Signaling by EpoR starts with JAK2 activation. Three EpoR cytoplasmic hydrophobic residues located before Box1 seem to be crucial for activating JAK2.14 These residues however are not required for the chaperone effect of JAK2 on EpoR trafficking. Upon receptor activation, JAK2 becomes phosphorylated at the activation loop Y1007, Y1008 and at many other Y residues, such as at Y570 (with inhibitory effect) and at Y221 and Y813 (with positive effects on signaling).16,17 JAK2 also phosphorylates the EpoR cytoplasmic Y residues. All these p-Y residues in EpoR and JAK2 become recruitment sites for STAT5, STAT3, STAT1, SH-PTP1, CIS and shc/grb2. As a consequence, EpoR signaling activates STAT, MAP-kinase, PI-3-kinase and AKT. The biological consequences of this cascade of events are survival, proliferation and differentiation of erythroid progenitors.

Several lines of evidence suggested that JAK2 was the most likely candidate gene involved in the pathogenesis of PV. First, JAK2 is directly involved in the intracellular signaling following the exposure to cytokines to which PV progenitors display hypersensitivity (Epo, GM-CSF, IL-3,TPO and more or less SCF and IGF-1). Second, spontaneous activation of STAT3 was found in the granulocytes of 30% of PV patients18 and an increased AKT phosphorylation was observed in erythroid progenitors after cytokine stimulation19 (Figure 1; see Color Figures, page 545). In erythroblasts derived from EEC, the anti-apoptotic mitochondrial protein Bcl-xL regulated by Epo signaling through STAT5 activation was shown to be increased.20 Moreover, different kinase inhibitors, including a JAK2 inhibitor are known to block EEC formation.21 Similarly in IMF, spontaneous MK growth was blocked by a STAT5 dominant negative.22 Third, JAK2 is involved in EpoR15 and Mpl23 trafficking from the endoplasmic reticulum to the cell membrane, and the processes involving Mpl trafficking/maturation are altered in PV and IMF.24 Fourth, although not recurrent, a common cytogenetic abnormality in PV is a gain in 9p where the JAK2 gene is localized. In addition, loss of heterozygosity (LOH) of chromosome 9p was found in 30% of PV patients identifying a large genomic region of DNA as a target for the search for a potential candidate disease associated genetic defect.25

It seems likely that some as yet unknown factors might modulate the activity of the mutant JAK2. The V617F substitution is a subtle mutation that only changes the basal JAK2 activation, otherwise all the biological properties of JAK2 are maintained including its binding to cytokine receptors. However, it is not known if the mutant JAK2 requires receptor binding or spontaneously oligomerizes on the membrane or in the cytosol to induce signaling. The highly transforming TEL-JAK2 protein that has lost all the regulatory and cytokine receptor elements of JAK2 induces direct signaling by oligomerization. Thus, it seems likely that slight changes in the V617F JAK2 kinase activity might profoundly change the phenotype of the disease. In favor of this hypothesis, the V617F mutation was found to be homozygous in about 30% of the PV, which correlates with the 9p LOH, which is due to a mitotic recombination and leads to a duplication of the mutated allele with a deletion of the normal allele.28,30 This implies that the cells homozygous for the mutation have a clonal advantage over the heterozygous and non-mutated cells.30 Usually, LOH is a mechanism of oncogenesis by which tumor suppressor genes are inactivated. To our knowledge, LOH has never been associated with gain of function mutations. Thus, gene dosage and the presence of a normal JAK2 may influence the activity of the mutant. Indeed, normal JAK2 is capable of inhibiting STAT5 activation induced by the mutant. Furthermore, expression of normal JAK2 in a cell line made factor-independent by the mutant restores cytokine dependency.26 The competition between the two JAK2s, wild type and mutated, is not directly due to inhibition of the mutant since wild type JAK2 does not inhibit auto-phosphorylation of the mutant28 but may occur on their substrate(s) by competing for cytokine receptor binding. One might hypothesize that the level of kinase activity modulates the phenotype of the disease. The level of kinase activity could be modified by gene dosage, by polymorphisms of JAK2 or by cooperating genetic events, such as changes in a phosphatase or a SOCS gene, which encode for proteins that normally attenuate JAK2 signaling from cytokine receptors. We hypothesize that a low kinase activity would lead to ET, an intermediate level to PV and a very high level to IMF. Alternatively, the phenotype of the disease may be dependent on genetic events not related to the V617F JAK2 kinase activity. These different hypotheses can now be tested both in patients and in animal models of MPD.

Luciferase signal was equivalent in MYC-luc;sg-p53, MYC-lucOS;sg-p53, MYC-luc;CTNNB1, and MYC-lucOS;CTNNB1 livers six days after the hydrodynamic injection (Supplementary Fig. S4D), excluding that differences in initial hepatocyte transfection could have an effect on the immune surveillance and immune escape observed in the MYC-lucOS;sg-p53 and MYC-lucOS;CTNNB1 mice, respectively. Furthermore, MYC-luc;sg-p53 and MYC-luc;CTNNB1 mice, which are not subjected to immune pressure, presented similar median survival [35 vs. 35.5 days in females (Figs. 1E and 4E); 44 vs. 42 days in males (Figs. 1F and 4F)], indicating that the tumor growth rate was similar in both models. As expected, MYC;CTNNB1 tumors overexpressed MYC, activated β-catenin, and displayed WT p53 (Supplementary Figs. S1E and S4E). To address whether β-catenin activation is truly driving immune escape of MYC-lucOS;CTNNB1 tumors and to rule out the potential involvement of WT p53, we assessed the effect of mutating p53 in the context of MYC-lucOS;CTNNB1 tumors. Tumor formation and survival were equivalent in MYC-lucOS;CTNNB1;sg-p53 and MYC-lucOS;CTNNB1 mice (Supplementary Fig. S4F and S4G), confirming the role of β-catenin activation in driving immune escape of MYC-lucOS;CTNNB1 tumors. These results also indicate that β-catenin activation can directly promote immune escape of MYC-lucOS;sg-p53 tumors, which otherwise undergo immune surveillance (Fig. 1).

We also assessed the transcriptional profiles of 360 samples from patients with HCC [liver hepatocellular carcinoma (LIHC), available at The Cancer Genome Atlas (TCGA); ref. 37]. As expected, CTNNB1-mutant samples (97/360, 26.79%) were significantly enriched in the dataset representing CTNNB1-mutant HCCs (36) when compared with CTNNB1 WT samples (Supplementary Table S2). In fact, expression of AXIN2 and GLUL, two well-established targets of β-catenin, was significantly higher in CTNNB1-mutant samples (Fig. 5H; Supplementary Table S4). Most importantly, CTNNB1-mutant samples presented significantly reduced expression of DC markers (BATF3, IRF8, THBD), T-cell markers (CD3D, CD3E, CD4, CD8A), and the exhaustion marker PDCD1 (PD-1; Fig. 5H; Supplementary Table S3), suggesting that CTNNB1-mutant HCCs exhibit immune exclusion. In fact, in a cohort of 59 HCC patient samples, nuclear staining of β-catenin was associated with significantly lower numbers of CD8+ T cells in the tumors (Fig. 5I and J), and in another cohort of 216 patients with HCC, those with enrichment of CTNNB1-mutant HCC signature (36) were associated with a significant decrease in immune cell infiltration assessed by hematoxylin and eosin staining (Supplementary Fig. S5L). To test the importance of β-catenin pathway activation levels on the immune escape phenotype, we stratified the 360 patients with HCC according to their level of enrichment of the dataset representing CTNNB1-mutant HCCs (ref. 36; low, first tertile; intermediate, second tertile; high, third tertile). As observed in the murine tumors (Fig. 5G; Supplementary Table S3), samples from patients with HCC with intermediate and high activation of the β-catenin pathway presented less immune cell transcripts than samples in the low activation group, further suggesting that β-catenin pathway activation levels have an impact on the extent of immune exclusion. Taken together, the immune escape driven by β-catenin activation is mediated by a defect in DC recruitment, which in turn impairs the subsequent antitumor immune response, in both murine and human HCCs. 2b1af7f3a8