SIRT1 Activation by a c-MYC Oncogenic Network Promotes the Maintenance and Drug Resistance of Human FLT3-ITD Acute Myeloid Leukemia Stem Cells

SummaryThe 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

T Cell Antigen Discovery Using Soluble Vaccinia Proteome Reveals Recognition of Antigens with Both Virion and Nonvirion Association

Vaccinia virus (VACV) is a useful model system for understanding the immune response to a complex pathogen. Proteome-wide Ab profiling studies reveal the humoral response to be strongly biased toward virion-associated Ags, and several membrane proteins induce Ab-mediated protection against VACV challenge in mice. Some studies have indicated that 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 strain Western Reserve (VACV-WR) plasmid expression library, produced previously for proteome microarrays for Ab profiling, to make a solubilized full VACV-WR proteome for T cell Ag 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 Ags 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 Abs from the same mice were enriched for membrane and other virion components, as described previously. We conclude that a "nonbiasing" approach to T cell Ag discovery reveals a T cell Ag profile in VACV that is broader and less skewed to virion association than the Ab profile. The T cell Ag mapping method developed in the present study should be applicable to other organisms where expressible "ORFeome" libraries are also available, and it is readily scalable for larger pathogens

Immunodominant "Asymptomatic" Herpes Simplex Virus 1 and 2 Protein Antigens Identified by Probing Whole-ORFome Microarrays with Serum Antibodies from Seropositive Asymptomatic versus Symptomatic Individuals

Herpes simplex virus 1 (HSV-1) and HSV-2 are medically significant pathogens. The development of an effective HSV vaccine remains a global public health priority. HSV-1 and HSV-2 immunodominant “asymptomatic” antigens (ID-A-Ags), which are strongly recognized by B and T cells from seropositive healthy asymptomatic individuals, may be critical to be included in an effective immunotherapeutic HSV vaccine. In contrast, immunodominant “symptomatic” antigens (ID-S-Ags) may exacerbate herpetic disease and therefore must be excluded from any HSV vaccine. In the present study, proteome microarrays of 88 HSV-1 and 84 HSV-2 open reading frames(ORFs) (ORFomes) were constructed and probed with sera from 32 HSV-1-, 6 HSV-2-, and 5 HSV-1/HSV-2-seropositive individuals and 47 seronegative healthy individuals (negative controls). The proteins detected in both HSV-1 and HSV-2 proteome microarrays were further classified according to their recognition by sera from HSV-seropositive clinically defined symptomatic (n = 10) and asymptomatic (n = 10) individuals. We found that (i) serum antibodies recognized an average of 6 ORFs per seropositive individual; (ii) the antibody responses to HSV antigens were diverse among HSV-1- and HSV-2-seropositive individuals; (iii) panels of 21 and 30 immunodominant antigens (ID-Ags) were identified from the HSV-1 and HSV-2 ORFomes, respectively, as being highly and frequently recognized by serum antibodies from seropositive individuals; and (iv) interestingly, four HSV-1 and HSV-2 cross-reactive asymptomatic ID-A-Ags, US4, US11, UL30, and UL42, were strongly and frequently recognized by sera from 10 of 10 asymptomatic patients but not by sera from 10 of 10 symptomatic patients (P < 0.001). In contrast, sera from symptomatic patients preferentially recognized the US10 ID-S-Ag (P < 0.001). We have identified previously unreported immunodominant HSV antigens, among which were 4 ID-A-Ags and 1 ID-S-Ag. These newly identified ID-A-Ags could lead to the development of an efficient “asymptomatic” vaccine against ocular, orofacial, and genital herpes

T Cell Antigen Discovery Using Soluble Vaccinia Proteome Reveals Recognition of Antigens with Both Virion and Nonvirion Association

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

Antibody Profiling by Proteome Microarray Reveals the Immunogenicity of the Attenuated Smallpox Vaccine Modified Vaccinia Virus Ankara Is Comparable to That of Dryvax▿ †

Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus that is under consideration as an alternative to the conventional smallpox vaccine Dryvax. MVA was attenuated by extensive passage of vaccinia virus Ankara in chicken embryo fibroblasts. Several immunomodulatory genes and genes that influence host range are deleted or mutated, and replication is aborted in the late stage of infection in most nonavian cells. The effect of these mutations on immunogenicity is not well understood. Since the structural genes appear to be intact in MVA, it is hypothesized that critical targets for antibody neutralization have been retained. To test this, we probed microarrays of the Western Reserve (WR) proteome with sera from humans and macaques after MVA and Dryvax vaccination. As most protein sequences of MVA are 97 to 99% identical to those of other vaccinia virus strains, extensive binding cross-reactivity is expected, except for those deleted or truncated. Despite different hosts and immunization regimens, the MVA and Dryvax antibody profiles were broadly similar, with antibodies against membrane and core proteins being the best conserved. The responses to nonstructural proteins were less well conserved, although these are not expected to influence virus neutralization. The broadest antibody response was obtained for hyperimmune rabbits with WR, which is pathogenic in rabbits. These data indicate that, despite the mutations and deletions in MVA, its overall immunogenicity is broadly comparable to that of Dryvax, particularly at the level of antibodies to membrane proteins. The work supports other information suggesting that MVA may be a useful alternative to Dryvax

An Extremely Diverse CD4 Response to Vaccinia Virus in Humans Is Revealed by Proteome-Wide T-Cell Profiling▿

CD4 T cells are required for the maintenance and recall of antiviral CD8 T cells and for antibody responses. Little is known concerning the overall architecture of the CD4 response to complex microbial pathogens. In a whole-proteome approach, 180 predicted open reading frames (ORFs) in the vaccinia virus genome were expressed and tested using responder cells from 20 blood samples from 11 vaccinees. Validation assays established the sensitivity and specificity of the system. Overall, CD4 responses were detected for 122 ORFs (68%). A mean of 39 ORFs were recognized per person (range, 13 to 63). The most frequently recognized ORFS were present in virions, including A3L and A10L (core proteins), WR148 (a fragmented homolog of an orthopoxvirus protein that forms inclusions in cells), H3L (a membrane protein), D13L (a membrane scaffold protein), and L4R (a nucleic acid binding protein). Serum immunoglobulin G profiling by proteome microarray detected responses to 45 (25%) of the ORFs and confirmed recent studies showing a diverse response directed to membrane and nonmembrane antigens. Our results provide the first empirical whole-proteome data set regarding the global CD4 response to full-length proteins in a complex virus and are consistent with the theory that abundant structural proteins are immunodominant

Principal component analysis of top antigens.

<p>Unsupervised principal component analysis of the signal intensity for samples from PV patients and healthy controls revealed that these two groups could be segregated on the basis of top 30 antigens with the highest sensitivity (<b>A</b>) and 15 antigens with the highest specificity (<b>B</b>) for PV.</p