Cancer-testis antigens (CTA), such as NY-ESO-1, MAGE-A1, and MAGE-A3, are 
immunogenic proteins encoded by genes, which are normally expressed only in male 
germ cells but are activated by ill-defined epigenetic mechanisms in human 
tumors, including lung cancers. Previously, we reported induction of these CTAs 
in cancer cells, but not normal cells, by DNA-demethylating agents and histone 
deacetylase inhibitors using clinically achievable exposure conditions. In the 
present study, we evaluated chromatin alterations associated with 
repression/activation of cancer-testis genes in lung cancer cells to further 
develop gene-induction regimens for cancer immunotherapy. Repression of 
NY-ESO-1, MAGE-A1, and MAGE-A3 coincided with DNA hypermethylation, recruitment, 
and binding of polycomb-group proteins, and histone heterochromatin 
modifications within the promoters of these genes. Derepression coincided with 
DNA demethylation, dissociation of polycomb proteins, and presence of 
euchromatin marks within the respective promoters. Short hairpin RNAs were used 
to inhibit several histone methyltransferases (KMT) and histone demethylases 
(KDM) that mediate histone methylation and repress gene expression. Knockdown of 
KMT6, KDM1, or KDM5B markedly enhanced deoxyazacytidine (DAC)-mediated 
activation of these cancer-testis genes in lung cancer cells. DZNep, a 
pharmacologic inhibitor of KMT6 expression, recapitulated the effects of KMT6 
knockdown. Following DAC-DZNep exposure, lung cancer cells were specifically 
recognized and lysed by allogeneic lymphocytes expressing recombinant T-cell 
receptors recognizing NY-ESO-1 and MAGE-A3. Combining DNA-demethylating agents 
with compounds, such as DZNep, that modulate histone lysine methylation may 
provide a novel epigenetic strategy to augment cancer-testis gene expression as 
an adjunct to adoptive cancer immunotherapy.