Strategies to overcome obstacles to successful immunotherapy of melanoma.

The immunogenicity of malignant melanomas has been recognized by the observed recruitment of tumor-specific cytotoxic T-cells (CTL), leading to the identification of several melanoma associated antigen (MAA). However, numerous strategies to treat melanoma with immunotherapy have resulted in only partial success. In this editorial, we discuss recent data related to the ability of tumors to elude immune responses. We therefore discuss different strategies to induce a clinically effective immune response. These approaches include 1) immunostimulation: including peptide/protein based vaccines, dendritic cell vaccines, and adoptive cell transfer; and 2) overcoming immunosuppression, including targeting of checkpoint molecules such as CTLA-4, circumventing the activity of Tregs, and assuring antigen expression by tumor cells (thwarting antigen silencing). Finally, we discuss recent advances in gene therapy, including adoptive therapy with engineered T cell receptors (TCRs). These issues lead to the conclusion that successful immunotherapy in malignant melanoma requires a combination of strategies aimed at both inducing immunostimulation and blocking immunosuppression.

Characterization of nucleophosmin (B23) as a Myc target by scanning chromatin immunoprecipitation.

The genetic program through which a specific transcription factor regulates a biological response is fundamental to our understanding how instructions in the genome are implemented. The emergence of DNA microarray technology for gene expression analysis has generated vast numbers of target genes resulting from specific transcription factor activity. We use the oncogenic transcription factor c-Myc as proof-of-principle that human genome sequence analysis and scanning of a specific gene by chromatin immunoprecipitation can be coupled to identify target transcription factor binding sequences. We focused on nucleophosmin, also known as B23, which was identified as a candidate Myc-responsive gene from a subtractive hybridization screen, and we found that sequences in intron 1, and not 5' sequences in the proximal promoter, are bound by c-Myc in vivo. Hence, a scanning chromatin immunoprecipitation (SChIP) strategy is useful in analyzing functional transcription factor-binding sites.

IF3-mediated suppression of a GUA initiation codon mutation in the recJ gene of Escherichia coli.

A mutational change of the initiation codon to GUA was found to reduce, but not abolish, expression of the recJ gene of Escherichia coli. Specific mutations in translational initiation factor IF3 have been isolated as second-site suppressors of this GUA initiation codon mutation. One of these, infC135, with an arginine-to-proline change at amino acid 131, completely restores a wild-type phenotype to recJ GUA initiation codon mutants and acts in a semidominant fashion. The infC135 mutation increased expression of RecJ from the GUA mutant but had no effect on the normal GUG start. The infC135 mutation also abolished autoregulation of IF3 in cis and in trans. The behavior of this IF3 mutant suggests that it has specifically lost its ability to abort initiation from poor initiation codons such as GUA of recJ and the AUU of infC. Because of the impact of IF3 on recJ, a recombination and repair gene, this role of IF3 must be general and not restricted to translation genes. The dominance of infC135 suggests that the other functions of IF3, for instance its ability to bind to 30S ribosomes, must remain intact. Although the ability to discriminate among initiation codons has been lost in the infC135 mutant, translational initiation was still restricted to the normal initiation site in recJ, even in the presence of a closely juxtaposed alternative initiation codon. Because the recJ gene lacks a canonical Shine-Dalgarno sequence, other unknown features of the mRNA must serve to specify the initiation site.

Suppression of recJ mutations of Escherichia coli by mutations in translation initiation factor IF3.

We have isolated genetic suppressors of mutations in the recJ gene of Escherichia coli in a locus we term srjA. These srjA mutations cause partial to complete alleviation of the recombination and UV repair defects conferred by recJ153 and recJ154 mutations in a recBC sbcA genetic background. The srjA gene was mapped to 37.5 min on the E. coli chromosome. This chromosomal region from the srjA5 strain was cloned into a plasmid vector and was shown to confer recJ suppression in a dominant fashion. Mutational analysis of this plasmid mapped srjA to the infC gene encoding translation initiation factor 3 (IF3). Sequence analysis revealed that all three srjA alleles cause amino acid substitutions of IF3. Suppression of recJ was shown to be allele specific: recJ153 and recJ154 mutations were suppressible, but recJ77 and the insertion allele recJ284::Tn10 were not. In addition, growth medium-conditional lethality was observed for strains carrying srjA mutations with the nonsuppressible recJ alleles. When introduced into recJ+ strains, srjA mutations conferred hyperrecombinational and hyper-UVr phenotypes. An interesting implication of these genetic properties of srjA suppression is that IF3 may regulate the expression of recJ and perhaps other recombination genes and hence may regulate the recombinational capacity of the cell.