Identifying Epigenetic and Mutational Barriers Limiting Cancer Cell Reprogramming Into Dendritic Cells

Cell reprogramming underscored a remarkable plasticity of somatic cells, opening avenues for regenerative medicine as well as cell replacement therapies. Through enforced expression of the three transcription factors (TFs) PU.1, IRF8 and BATF3, our group demonstrated that fibroblasts can be reprogrammed into conventional type 1 dendritic cells (cDC1) fate. Furthermore, we showed that the same combination of TFs can be applied to reprogram cancer cells, generating tumor-antigen presenting cells (tumor-APCs) with highly immunogenic features and functional capacity to restore anti-cancer immune response. However, the efficiency of reprogramming varied significantly among different cancer cell lines. We hypothesized that the efficiency of cancer cell reprogramming into cDC1 might be limited by preexisting epigenetic and genetic barriers. Here, we investigated the impact of chromatin-based barriers on cDC1 reprogramming by removing repressive marks using histone deacetylase inhibitor valproic acid (VPA) and inhibitor of DNA demethylases, 5-azacytidine (AZA) during reprogramming in 26 various human cancer cell lines. Overall, VPA had a superior effect on increasing reprogramming efficiency compared to treatment with AZA, suggesting that histone deacetylation represents a more frequent barrier in reprogramming. Although AZA showed a generally lower increase in reprogramming efficiency, it revealed that DNA methylation is also a limiting barrier in reprogramming, particularly in certain cancer cell lines. Mechanistically, we showed that treatment with VPA accelerates the PIB-mediated reprogramming process, leading to faster acquisition of a mature phenotype in reprogrammed tumor-APCs. Nevertheless, the majority of tested cell lines only showed partial improvement in reprogramming efficiency in response to treatment with epigenetic modifiers, suggesting preexisting genetic barriers (mutations) hindering reprogramming. By integrating mutational profiles of cell lines identified by whole exome sequencing with the epigenetic analysis, we predicted mutations that negatively affect reprogramming efficiencies and cannot be reverted by epigenetic modulation. We identified mutations in 6 genes (BPIFB6, BPIFB3, ITGA4, OR5H15, KRTAP10-6, and SPATA22) that correlated with impaired reprogramming. Although the identified mutations do not affect cDC1 signature genes, they may play a role in cellular plasticity and maintenance of identity in cells. Validating these mutations and assessing their impact on reprogramming efficiency and functionality of reprogrammed cells is crucial for further understanding of their role. To this end, understanding the factors limiting cell reprogramming is of key importance to improve the efficacy of cDC1 reprogramming and advancing its translation into cancer immunotherapy, as well as improving the efficiency of other reprogramming-based strategies.