Intratumoral Heterogeneity in Cancer/testis Antigen Expression in Solid Tumors, Implications for Cancer Immunotherapy

Epigenic alterations, including changes in DNA methylation, are fundamental characteristics of cancer cells. These cancer-related changes in methylation patterns can result in genomic instability, increased expression of antigens typically not expressed in healthy somatic tissue, such as cancer/testis antigens (CTAs), and decreased expression of tumor suppressor genes. Due to their frequent and specific expression in cancer cells, CTAs are considered promising targets for immunotherapeutic treatment of solid cancers. However, the variability in CTA expression among different tumors and even between cancer cells within the same tumor can lead to escape of CTA-negative cancer cells when targeting these antigens with immunotherapy. Treatment with DNA methyltransferase inhibitors (DNMTis) has been proposed to increase CTA expression among cancer cells, potentially enhancing the effectiveness of CTA-directed immunotherapies. Further, DNMTi monotherapy has shown efficacy in treating hematological cancers, however, this effectiveness has not been expanded to treatment of solid cancers.

The aim of my project was to further explore the potential of CTAs as targets for immunotherapeutic treatment of solid cancers and to investigate the use of DNMTi treatment for upregulation of CTA expression and as a monotherapy for solid cancers. We examined the expression of the CTA VCX2 in various types of normal and malignant Qssues and found that VCX2 was only expressed in a small number of melanoma tumors and that VCX2 expression was highly tumor specific. Furthermore, VCX2 expression could be upregulated by treatment with a DNMTis and further augmented by combination treatment with DNMTis and histone deacetylase inhibitors (HDACis). However, VCX2 alone may not be suitable as a target for immunotherapy; instead, it could be included in a panel of CTAs for multitargeting immunotherapies, such as vaccines, for the treatment of VCX2-posiQve melanoma tumors. Additionally, targeted single-cell mRNA sequencing (scRNAseq) of primary melanoma cell cultures and cells from fresh lung adenocarcinomas, revealed a previously unrecognized variability in CTA expression within these cancer types, which could have implications for the development of future immunotherapeutic strategies targeting these antigens. Homogeneous expression of individual CTAs was rare, observed only in melanoma, emphasizing the importance of immunotherapeutic approaches targeting multiple CTAs to prevent evasion by CTA-negative subclones. Furthermore, scRNAseq of breast cancer cell lines demonstrated a highly variable response to DNMTi treatment, which could be mitigated by combination treatment with DNMTis and HDACis. This suggests that this combination treatment could enhance the clinical effectiveness of epigenetic treatment of solid cancers and minimize variability in CTA expression, thereby improving the efficacy of CTA-directed immunotherapies. Our results underscore the importance of characterizing patient tumors at the single-cell level to select appropriate CTA targets for personalized immunotherapeutic treatment of patients with solid tumors.