Extended long-term follow-up of metastatic melanoma patients treated with immunotherapy: late relapses and second primary melanomas.

Background: Immunotherapy has revolutionized the treatment of patients with advanced melanoma as well as other cancers. Most studies, whether of interleukin-2 or checkpoint inhibitor therapies, have limited follow-up after 5 years, making the incidence of late relapses uncertain. In addition, the incidence of second primary melanomas in patients with stage IV melanoma treated with immunotherapy has rarely been reported. Methods: We performed a single-institution retrospective study of stage IV melanoma patients treated with interleukin-2 or checkpoint inhibitors over the period from 1992 to 2013. We found 59 patients alive and in remission 5 years after the beginning of immunotherapy and reviewed their subsequent clinical course. Results: This 59-patient cohort had a median follow-up of 13.1 years, with 36 patients followed up for at least 10 years. Four patients (6.8%) had relapses of their metastatic melanoma at 5, 8, 15, and 17 years after starting the successful immunotherapy. Three of the four are still alive. Only one patient in 690 patient-years of observation had a second primary invasive melanoma. Conclusion: Although late relapses after immunotherapy for melanoma do occur, we can conclude that the prognosis of stage IV melanoma patients in continuous remission 5 years after starting immunotherapy is excellent, with a progression-free survival of approximately 85% and a melanoma-specific survival of approximately 95% at 20 years in our series. Our incidence of second primary melanomas is lower than usually reported. These results have important implications regarding the follow-up of stage IV melanoma patients successfully treated with immunotherapy.

NetAct: a computational platform to construct core transcription factor regulatory networks using gene activity.

A major question in systems biology is how to identify the core gene regulatory circuit that governs the decision-making of a biological process. Here, we develop a computational platform, named NetAct, for constructing core transcription factor regulatory networks using both transcriptomics data and literature-based transcription factor-target databases. NetAct robustly infers regulators' activity using target expression, constructs networks based on transcriptional activity, and integrates mathematical modeling for validation. Our in silico benchmark test shows that NetAct outperforms existing algorithms in inferring transcriptional activity and gene networks. We illustrate the application of NetAct to model networks driving TGF-ß-induced epithelial-mesenchymal transition and macrophage polarization.

Deconvolution of chromatin immunoprecipitation-microarray (ChIP-chip) analysis of MBF occupancies reveals the temporal recruitment of Rep2 at the MBF target genes.

MBF (or DSC1) is known to regulate transcription of a set of G(1)/S-phase genes encoding proteins involved in regulation of DNA replication. Previous studies have shown that MBF binds not only the promoter of G(1)/S-phase genes, but also the constitutive genes; however, it was unclear if the MBF bindings at the G(1)/S-phase and constitutive genes were mechanistically distinguishable. Here, we report a chromatin immunoprecipitation-microarray (ChIP-chip) analysis of MBF binding in the Schizosaccharomyces pombe genome using high-resolution genome tiling microarrays. ChIP-chip analysis indicates that the majority of the MBF occupancies are located at the intragenic regions. Deconvolution analysis using Rpb1 ChIP-chip results distinguishes the Cdc10 bindings at the Rpb1-poor loci (promoters) from those at the Rpb1-rich loci (intragenic sequences). Importantly, Res1 binding at the Rpb1-poor loci, but not at the Rpb1-rich loci, is dependent on the Cdc10 function, suggesting a distinct binding mechanism. Most Cdc10 promoter bindings at the Rpb1-poor loci are associated with the G(1)/S-phase genes. While Res1 or Res2 is found at both the Cdc10 promoter and intragenic binding sites, Rep2 appears to be absent at the Cdc10 promoter binding sites but present at the intragenic sites. Time course ChIP-chip analysis demonstrates that Rep2 is temporally accumulated at the coding region of the MBF target genes, resembling the RNAP-II occupancies. Taken together, our results show that deconvolution analysis of Cdc10 occupancies refines the functional subset of genomic binding sites. We propose that the MBF activator Rep2 plays a role in mediating the cell cycle-specific transcription through the recruitment of RNAP-II to the MBF-bound G(1)/S-phase genes.

Genomic binding profiling of the fission yeast stress-activated MAPK Sty1 and the bZIP transcriptional activator Atf1 in response to H2O2.

BACKGROUND: The evolutionally conserved MAPK Sty1 and bZIP transcriptional activator Atf1 are known to play a pivotal role in response to the reactive oxygen species in S. pombe. However, it is unclear whether all of the H(2)O(2)-induced genes are directly regulated by the Sty1-Atf1 pathway and involved in growth fitness under H(2)O(2)-induced stress conditions. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the study on ChIP-chip mapping of the genomic binding sites for Sty1, Atf1, and the Atf1's binding partner Pcr1; the genome-wide transcriptional profiling of the atf1 and pcr1 strains in response to H(2)O(2); and the phenotypic assessment of approximately 90 Atf1/Pcr1-bound or unbound genes for growth fitness under H(2)O(2) conditions. ChIP-chip analysis shows that Atf1 and Pcr1 binding sites are overlapped in the genome and constitutively present before H(2)O(2) stress. On the other hand, Sty1 recruitment primarily occurs at the Atf1/Pcr1 binding sites and is induced by H(2)O(2). We found that Atf1/Pcr1 is clearly responsible for the high-level transcriptional response to H(2)O(2). Furthermore, phenotypic assessment indicates that among the H(2)O(2)-induced genes, Atf1/Pcr1-bound genes exhibit a higher likelihood of functional requirement for growth fitness under the stress condition than the Atf1/Pcr1-unbound genes do. Notably, we found that the Atf1/Pcr1-bound genes regardless of their responsiveness to H(2)O(2) show a high probability of requirement for growth fitness. CONCLUSION/SIGNIFICANCE: Together, our analyses on global mapping of protein binding sites, genome-wide transcriptional profiling, and phenotypic assessment provide insight into mechanisms for global transcriptional regulation by the Sty1-Atf1 pathway in response to H(2)O(2)-induced reactive oxygen species.

The core and accessory genomes of Burkholderia pseudomallei: implications for human melioidosis.

Natural isolates of Burkholderia pseudomallei (Bp), the causative agent of melioidosis, can exhibit significant ecological flexibility that is likely reflective of a dynamic genome. Using whole-genome Bp microarrays, we examined patterns of gene presence and absence across 94 South East Asian strains isolated from a variety of clinical, environmental, or animal sources. 86% of the Bp K96243 reference genome was common to all the strains representing the Bp "core genome", comprising genes largely involved in essential functions (eg amino acid metabolism, protein translation). In contrast, 14% of the K96243 genome was variably present across the isolates. This Bp accessory genome encompassed multiple genomic islands (GIs), paralogous genes, and insertions/deletions, including three distinct lipopolysaccharide (LPS)-related gene clusters. Strikingly, strains recovered from cases of human melioidosis clustered on a tree based on accessory gene content, and were significantly more likely to harbor certain GIs compared to animal and environmental isolates. Consistent with the inference that the GIs may contribute to pathogenesis, experimental mutation of BPSS2053, a GI gene, reduced microbial adherence to human epithelial cells. Our results suggest that the Bp accessory genome is likely to play an important role in microbial adaptation and virulence.

Global profiling of DNA replication timing and efficiency reveals that efficient replication/firing occurs late during S-phase in S. pombe.

BACKGROUND: During S. pombe S-phase, initiation of DNA replication occurs at multiple sites (origins) that are enriched with AT-rich sequences, at various times. Current studies of genome-wide DNA replication profiles have focused on the DNA replication timing and origin location. However, the replication and/or firing efficiency of the individual origins on the genomic scale remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Using the genome-wide ORF-specific DNA microarray analysis, we show that in S. pombe, individual origins fire with varying efficiencies and at different times during S-phase. The increase in DNA copy number plotted as a function of time is approximated to the near-sigmoidal model, when considering the replication start and end timings at individual loci in cells released from HU-arrest. Replication efficiencies differ from origin to origin, depending on the origin's firing efficiency. We have found that DNA replication is inefficient early in S-phase, due to inefficient firing at origins. Efficient replication occurs later, attributed to efficient but late-firing origins. Furthermore, profiles of replication timing in cds1Delta cells are abnormal, due to the failure in resuming replication at the collapsed forks. The majority of the inefficient origins, but not the efficient ones, are found to fire in cds1Delta cells after HU removal, owing to the firing at the remaining unused (inefficient) origins during HU treatment. CONCLUSIONS/SIGNIFICANCE: Taken together, our results indicate that efficient DNA replication/firing occurs late in S-phase progression in cells after HU removal, due to efficient late-firing origins. Additionally, checkpoint kinase Cds1p is required for maintaining the efficient replication/firing late in S-phase. We further propose that efficient late-firing origins are essential for ensuring completion of DNA duplication by the end of S-phase.

Modulation of cell cycle-specific gene expressions at the onset of S phase arrest contributes to the robust DNA replication checkpoint response in fission yeast.

Fission yeast replication checkpoint kinases Rad3p and Cds1p are essential for maintaining cell viability after transient treatment with hydroxyurea (HU), an agent that blocks DNA replication. Although current studies have focused on the cyclin-dependent protein kinase Cdc2p that is regulated by these checkpoint kinases, other aspects of their functions at the onset of S phase arrest have not been fully understood. In this study, we use genome-wide DNA microarray analyses to show that HU-induced change of expression profiles in synchronized G(2) cells occurs specifically at the onset of S phase arrest. Induction of many core environmental stress response genes and repression of ribosomal genes happen during S phase arrest. Significantly, peak expression level of the MluI-like cell cycle box (MCB)-cluster (G(1)) genes is maintained at the onset of S phase arrest in a Rad3p- and Cds1p-dependent manner. Expression level maintenance of the MCB-cluster is mediated through the accumulation of Rep2p, a putative transcriptional activator of the MBF complex. Conversely, the FKH-cluster (M) genes are repressed during the onset of S phase arrest in a Rad3p-dependent manner. Repression of the FKH-cluster genes is mediated through the decreased levels of one of the putative forkhead transcription factors, Sep1p, but not Fkh2p. Together, our results demonstrate that Rad3p and Cds1p modulate transcriptional response during the onset of S phase arrest.

Global map of growth-regulated gene expression in Burkholderia pseudomallei, the causative agent of melioidosis.

Many microbial pathogens express specific virulence traits at distinct growth phases. To understand the molecular pathways linking bacterial growth to pathogenicity, we have characterized the growth transcriptome of Burkholderia pseudomallei, the causative agent of melioidosis. Using a fine-scale sampling approach, we found approximately 17% of all B. pseudomallei genes displaying regulated expression during growth in rich medium, occurring as broad waves of functionally coherent gene expression tightly associated with distinct growth phases and transition points. We observed regulation of virulence genes across all growth phases and identified serC as a potentially new virulence factor by virtue of its coexpression with other early-phase virulence genes. serC-disrupted B. pseudomallei strains were serine auxotrophs and in mouse infection assays exhibited a dramatic attenuation of virulence compared to wild-type B. pseudomallei. Immunization of mice with serC-disrupted B. pseudomallei also conferred protection against subsequent challenges with different wild-type B. pseudomallei strains. At a genomic level, early-phase genes were preferentially localized on chromosome 1, while stationary-phase genes were significantly biased towards chromosome 2. We detected a significant level of chromosomally clustered gene expression, allowing us to predict approximately 100 potential operons in the B. pseudomallei genome. We computationally and experimentally validated these operons by showing that genes in these regions are preferentially transcribed in the same 5'-->3' direction, possess significantly shorter intergenic lengths than the overall genome, and are expressed as a common mRNA transcript. The availability of this transcriptome map provides an important resource for understanding the transcriptional architecture of B. pseudomallei.