The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and is associated with poor prognosis. In such patients, FLT3 tyrosine kinase inhibitors (TKIs) are only partially ...effective and do not eliminate the leukemia stem cells (LSCs) that are assumed to be the source of treatment failure. Here, we show that the NAD-dependent SIRT1 deacetylase is selectively overexpressed in primary human FLT3-ITD AML LSCs. This SIRT1 overexpression is related to enhanced expression of the USP22 deubiquitinase induced by c-MYC, leading to reduced SIRT1 ubiquitination and enhanced stability. Inhibition of SIRT1 expression or activity reduced the growth of FLT3-ITD AML LSCs and significantly enhanced TKI-mediated killing of the cells. Therefore, these results identify a c-MYC-related network that enhances SIRT1 protein expression in human FLT3-ITD AML LSCs and contributes to their maintenance. Inhibition of this oncogenic network could be an attractive approach for targeting FLT3-ITD AML LSCs to improve treatment outcomes.
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•SIRT1 protein expression is selectively enhanced in FLT3-ITD AML stem cells•Combined SIRT1 and FLT3 inhibition increases elimination of FLT3-ITD AML stem cells•SIRT1 overexpression in FLT3-ITD AML is related to c-MYC activation•c-MYC increases SIRT1 expression by enhancing expression of the USP22 deubiquitinase
Li et al. find that c-MYC via USP22 deubiquitinase enhances SIRT1 expression and promotes maintenance of human AML stem cells, which suggests a strategy to target leukemia stem cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Pemphigus vulgaris (PV) is a mucocutaneous blistering disease characterized by IgG autoantibodies against the stratified squamous epithelium. Current understanding of PV pathophysiology does not ...explain the mechanism of acantholysis in patients lacking desmoglein antibodies, which justifies a search for novel targets of pemphigus autoimmunity. We tested 264 pemphigus and 138 normal control sera on the multiplexed protein array platform containing 701 human genes encompassing many known keratinocyte cell-surface molecules and members of protein families targeted by organ-non-specific PV antibodies. The top 10 antigens recognized by the majority of test patients' sera were proteins encoded by the DSC1, DSC3, ATP2C1, PKP3, CHRM3, COL21A1, ANXA8L1, CD88 and CHRNE genes. The most common combinations of target antigens included at least one of the adhesion molecules DSC1, DSC3 or PKP3 and/or the acetylcholine receptor CHRM3 or CHRNE with or without the MHC class II antigen DRA. To identify the PV antibodies most specific to the disease process, we sorted the data based on the ratio of patient to control frequencies of antigen recognition. The frequency of antigen recognition by patients that exceeded that of control by 10 and more times were the molecules encoded by the CD33, GP1BA, CHRND, SLC36A4, CD1B, CD32, CDH8, CDH9, PMP22 and HLA-E genes as well as mitochondrial proteins encoded by the NDUFS1, CYB5B, SOD2, PDHA1 and FH genes. The highest specificity to PV showed combinations of autoantibodies to the calcium pump encoded by ATP2C1 with C5a receptor plus DSC1 or DSC3 or HLA-DRA. The results identified new targets of pemphigus autoimmunity. Novel autoantibody signatures may help explain individual variations in disease severity and treatment response, and serve as sensitive and specific biomarkers for new diagnostic assays in PV patients.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Highlights ► Vaccinia antibodies were profiled before and after MVA and DVX vaccination. ► As a result, WR148, D8L, D13L, H3L, A26L and I1L were purified and evaluated in ELISAs. ► The membrane ...protein, D8L, provided the best sensitivity and specificity for monitoring both vaccines. ► The A-type inclusion protein homolog, WR148, provided the best discrimination. ► A13L/I1L ratio discriminated primary and secondary responses to DVX.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Abstract 1368
In acute myeloid leukemia (AML) small populations of leukemia stem and progenitor cells are resistant to available therapies and represent an important barrier to response and cure. ...There is pressing need to develop new treatment approaches to target AML stem/progenitor cells. The NAD-dependent deacetylase SIRT1 plays an important role in protection of stem cells from stress. We have recently shown that SIRT1 inhibition enhances elimination of CML LSC via acetylation and activation of p53 (Cancer Cell 2012, 21:266). Here we evaluated the role of SIRT1 in growth and maintenance of AML stem/progenitor cells. We observed higher levels of SIRT1 expression in AML compared to normal CD34+CD38− and CD34+CD38+ cell, with highest expression levels in samples from patients with poor-risk cytogenetic abnormalities. We tested the effects of the SIRT1 inhibitor Tenovin-6 (TV-6) on CD34+ cells from high-risk AML patients (n=12) and healthy controls (n=4). AML CD34+ cells demonstrated varying degrees of sensitivity to TV-6, but TV-6 treatment did not significantly reduce viability of AML CD34+ cells taken as a group, compared to normal cells (p=0.8). However, we observed significantly reduced viability of a subset of samples bearing the FLT3-ITD mutation following TV-6 treatment (p=0.01). We therefore tested the effects of TV-6 on a larger set of poor risk AML samples with the FLT3-ITD mutation (n=11), or wild-type FLT3 (FLT3-WT, n=11). The FLT3-ITD mutation was associated with significantly enhanced sensitivity of AML CD34+ cells to TV-6 induced apoptosis (LD50 for FLT3-ITD=1.9μM; FLT3-WT=3.9μM, p=0.009), and inhibition of cell growth measured using the CellTitre Glo luminescent cell viability assay (FLT3-ITD IC50=0.73μM; versus FLT3-WT IC50=1.4μM, p=0.01). Therefore the presence of the FLT3-ITD mutation identifies a subset of AML samples that are very sensitive to SIRT1 inhibition. Tenovin-6 treatment increased acetylated as well as total p53 levels in FLT3-ITD+ AML CD34+ cells. Tenovin-6 also induced apoptosis and inhibited growth of the FLT3-ITD+ and p53 wild type cell line MV4-11. SIRT1 knock-down reduced TV-6-induced apoptosis and growth inhibition in MV4-11 cells confirming that TV-6 effects are related to SIRT1 inhibition. The growth inhibitory and pro-apoptotic effects of TV-6 in MV4-11 cells were also significantly reduced by p53 knock-down (TV-6 1μM, p53 shRNA 39±8% inhibition, control ShRNA 66±2% inhibition, p=0.04, n=3). Taken together these results support a potential role for p53 activation in mediating the effects of TV-6 treatment in FLT3-ITD+ AML cells. Sequencing studies indicated that only one sample in the FLT3-WT group harbored a p53 mutation, suggesting that their resistance to SIRT1 inhibitors is related to mechanisms other than p53 mutation. Flow cytometry analysis demonstrated increased SIRT1 levels in FLT3-ITD (n=7) compared to FLT3-WT (n=9) AML CD34+ cells (p=0.01). We used lentivirus vectors to ectopically express a FLT3-ITD construct in human cord blood CD34+ cells. Expression of FLT3-ITD resulted in significantly increased SIRT1 expression levels compared to FLT3-WT or vector controls. Consistent with results obtained with samples from AML patients, FLT3-ITD transformed CB CD34+ cells demonstrated enhanced sensitivity to TV-6 mediated apoptosis (TV6 1μM, FLT3-ITD 44±3% survival, n=5; FLT3-WT 59±2% survival, n=5, p=0.04; empty vector 64±4% survival, p=0.02, n=5) and cell growth inhibition. These results indicate that FLT3-ITD-mediated SIRT1 overexpression may contribute to the increased sensitivity of FLT3-ITD AML CD34+ cells to SIRT1 inhibition. In conclusion, our studies indicate that AML stem/progenitor cells demonstrate varying degrees of sensitivity to SIRT1 inhibition reflecting underlying genetic lesions. Importantly the poor prognosis FLT3-ITD mutation is associated with increased SIRT1 expression and enhanced sensitivity to SIRT1 inhibition. In view of the limited success of small molecule FLT3 inhibitors so far, the use of SIRT1 inhibitors may offer an attractive approach to target FLT3-ITD+ AML stem/progenitor cells.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Vaccinia virus (VACV) is a useful model system for understanding the immune response to a complex pathogen. Proteome-wide antibody profiling studies reveal the humoral response to be strongly biased ...towards virion associated antigens, and several membrane proteins induce antibody-mediated protection against VACV challenge in mice. Some studies have indicated the CD4 response is also skewed toward proteins with virion association, whereas the CD8 response is more biased toward proteins with early expression. In this study, we have leveraged a VACV-WR plasmid expression library, produced previously for proteome microarrays for antibody profiling, to make a solubilized full VACV-WR proteome for T cell antigen profiling. Splenocytes from VACV-WR-infected mice were assayed without prior expansion against the soluble proteome in assays for Th1 and Th2 signature cytokines. The response to infection was polarized toward a Th1 response, with the distribution of reactive T cell antigens comprising both early and late VACV proteins. Interestingly, the proportions of different functional subsets were similar to that present in the whole proteome. In contrast, the targets of antibodies from the same mice were enriched for membrane and other virion components, as described previously. We conclude that a ‘non-biasing’ approach to T cell antigen discovery reveals a T cell antigen profile in VACV that is broader and less skewed to virion-association than the antibody profile. The T cell antigen mapping method developed here should be applicable to other organisms where expressible ‘ORFeome’ libraries are also available, and is readily scalable for larger pathogens.
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