Scant information is available about the molecular basis of multiple HLA class I antigen-processing machinery defects in malignant cells, although this information contributes to our understanding of the molecular immunoescape mechanisms utilized by tumor cells and may suggest strategies to counteract them. In the present study we reveal a combination of IFN-γ-irreversible structural and epigenetic defects in HLA class I antigen-processing machinery in a recurrent melanoma metastasis after immunotherapy. These defects include loss of tapasin and one HLA haplotype as well as selective silencing of HLA-A3 gene responsiveness to IFN-γ. Tapasin loss is caused by a germ-line frameshift mutation in exon 3 (TAPBP^684delA) along with a somatic loss of the other gene copy. Selective silencing of HLA-A3 gene and its IFN-γ responsiveness is associated with promoter CpG methylation nearby site-α and TATA box, reversible after DNA methyltransferase 1 depletion. This treatment combined with tapasin reconstitution and IFN-γ stimulation restored the highest level of HLA class I expression and its ability to elicit cytotoxic T cell responses. These results represent a novel tumor immune evasion mechanism through impairing multiple components at various levels in the HLA class I antigen presentation pathway. These findings may suggest a rational design of combinatorial cancer immunotherapy harnessing DNA demethylation and IFN-γ response.

Anaplastic thyroid carcinoma (ATC) is almost universally fatal. Elevated keratin-8 (KRT8) protein expression is an established diagnostic cancer biomarker in several epithelial cancers (but not ATC). Several keratins, including KRT8, have been suggested to have a role in cell biology beyond that of structural cytoskeletal proteins. Here, we provide evidence that KRT8 plays a direct role in the growth of ATCs. Genomic and transcriptomic analysis of >5000 patients demonstrates that KRT8 mutation and copy number amplification are frequently evident in epithelial-derived cancers. Carcinomas arising from diverse tissues exhibit KRT8 mRNA and protein overexpression when compared to normal tissue levels. Similarly, in a panel of patient-derived ATC cell lines and patient tumors, KRT8 expression shows a similar pattern. sh-RNA-mediated KRT8 knockdown in these cell lines increases apoptosis, whereas forced overexpression of KRT8 confers resistance to apoptosis under peroxide-induced cell stress conditions. We further show that KRT8 protein binds to annexin A2, a protein known to mediate apoptosis as well as the redox pathway.

Limited information is currently available concerning HLA class I antigen abnormalities in sarcomatoid hepatocellular carcinoma (sHCC). Here, we have analyzed the growth characteristics and HLA class I antigen status of four sHCC cell lines (sHCC29, sHCC63, sHCC74, and SAR-HCV); the first three were newly established in this study. Among the four, sHCC29 showed the highest growth ratein vitroand tumorigenicity in NOD-SCID mice. Unlike sHCC74 and SAR-HCV, both sHCC29 and sHCC63 had no detectable surface HLA class I antigen expression, alongside undetected intracellularβ₂-microglobulin (β₂m) and marked HLA class I heavy chain and selective antigen-processing machinery (APM) component downregulation. The loss ofβ₂m in sHCC29 and sHCC63 was caused by a >49 kb deletion across theB2Mlocus, while their downregulation of APM components was transcriptional, reversible by IFN-γonly in several components.β₂m was also undetected in the primary HCC lesions of the patients involved, indicating itsin vivorelevance. We report for the first time HLA class I antigen loss with underlyingB2Mgene deficiency and APM defects in 50% (2 of 4) of the sHCC cell lines tested. These findings may have implications for a proper design of T cell immunotherapy for the treatment of sHCC patients.

The unsatisfactory real-world efficacy of the hypomethylating agent azacitidine in treating myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has prompted us to investigate the hematological adverse events and host variables that may compromise the use of this epigenetic drug. Using the zebrafish, we found that azacitidine destroyed their myeloid precursors and impaired myeloid function by inhibiting antigen processing, allogeneic response and phagocytic activity, resulting in increased susceptibility to infection even by the normal flora E. coli. In addition, iron overload, a MDS-associated condition following repeated transfusions, exacerbated bacterial infection especially by V. vulnificus with known iron dependence. Furthermore, we show that the tp53^M214K mutant zebrafish survived longer than the wild-type (WT) when challenged with bacteria following azacitidine treatment. This was attributed to the mutant’s hematopoietic cells rather than its general genetic background, since the WT animals reconstituted with the tp53^M214K mutant kidney marrow became more resistant to bacterial infection following treatment with azacitidine. The clinical relevance of our findings was indicated by a MDS case with severe azacitidine-induced bone marrow suppression and by the association of hyperferritinemia with bacteremia in azacitidine-treated patients, while tp53^M214K-mediated resistance to azacitidine-induced myelosuppression may explain the survival advantage of malignant MDS and AML clones over their normal counterparts under azacitidine treatment. Together, we propose that myelosuppression, iron overload and TP53 mutations may represent the host variables that compromise the azacitidine efficacy.