Chromatin regulation is a major aspect of cancer development, progression, and treatment. Several small molecule inhibitors of chromatin regulators are currently used for treatment of certain hematological malignancies. However, there is still opportunity for many more patients to benefit from therapeutic approaches that target chromatin regulation, especially in the context of solid tumors. A critical unmet need is the identification of robust biomarkers that can guide the application of epigenetic inhibitors in a precise and personalized manner. In my dissertation, I aim to address this important knowledge gap by studying how perturbation of chromatin can target metabolic and lineage abnormalities in solid tumors for therapeutic benefit. To do this, I have focused on genetic and pharmacological perturbations of chromatin pathways in two cancer models: (1) lung adenocarcinoma (LUAD) with NRF2 activation and (2) neuroendocrine esophageal carcinoma (NEC). In the study on NRF2-active LUAD, we found that histone deacetylase (HDAC) inhibitors can be repurposed to reprogram the epigenomic and metabolic landscape, which leads to specific and potent anti-tumor effects in the context of NRF2 activation. Specifically, we employed a chromatin-focused genetic screen to identify dependencies on chromatin regulators. The screen revealed an NRF2-specific dependency on class I histone deacetylases. Experiments in mouse and human LUAD cell lines in vitro and in vivo indicated an NRF2-specific sensitivity to the class I HDAC inhibitor Romidepsin. Mechanistically, profiling of histone acetylation and gene expression upon Romidepsin treatment revealed a relative loss of histone H4 acetylation at promoters which was associated with reduced gene expression. Many downregulated genes were more essential for the survival of NRF2 hyperactive cancer cells, including genes involved in glutamine and serine metabolism, c-Myc and several of its targets involved in purine and pyrimidine synthesis. These transcriptional changes had corresponding effects on altering the metabolic pathways that NRF2-active cells selectively require for survival. In the study on neuroendocrine esophageal carcinoma (NEC), we identified a crucial role for epigenetic regulation of lineage fate through transcriptional control of the key epidermal transcription factor p63. This project originated from data from my collaborators that indicates a role for p63 in the suppression of basal-to-neuroendocrine identity transition in the developing esophagus. Consistently, I found that p63 is silenced in NEC through a non-genetic mechanism. Reintroducing p63 isoforms in a human NEC cell line showed that ΔNp63α was sufficient to restore squamous marker expression. An epigenetic drug screen assessing p63 gene expression and subsequent validation experiments revealed that inhibition of EZH2, a histone methyltransferase, induced expression of ΔNp63α and genes related to the squamous identity. Analysis of the chromatin state in the TP63 locus showed that EZH2 inhibition led to a loss histone H3 methylation and a gain of histone H3 acetylation and its reader BRD4. These results support the hypothesis that the squamous identity can be reactivated epigenetically in NEC through de-repression of ΔNp63α as a potential therapeutic strategy. Together, these studies contribute to our understanding of the transcriptional response to chromatin perturbation and show that this can be leveraged to modulate cell metabolism and identity, as well as to achieve therapeutic benefit in new contexts of cancer.

The azanucleosides decitabine and azacytidine are used widely in the treatment of myeloid neoplasia and increasingly in the context of combination therapies. Although they were long regarded as being largely interchangeable in their function as hypomethylating agents, the azanucleosides actually have different mechanisms of action; decitabine interferes primarily with the methylation of DNA and azacytidine with that of RNA. Here, we examine the role of DNA methylation in the lineage commitment of stem cells during normal hematopoiesis and consider how mutations in epigenetic regulators such as DNMT3A and TET2 can lead to clonal expansion and subsequent neoplastic progression. We also consider why the efficacy of azanucleoside treatment is not limited to neoplasias carrying mutations in epigenetic regulators. Finally, we summarise recent data describing a role for azacytidine-sensitive RNA methylation in lineage commitment and in the cellular response to stress. By summarising and interpreting evidence for azanucleoside involvement in a range of cellular processes, our review is intended to illustrate the need to consider multiple modes of action in the design and stratification of future combination therapies.

The CpG Island Methylator Phenotype (CIMP) is a distinct form of aberrant DNA methylation in cancer, and it is seen in 20-40% of colorectal cancers (CRC) where its causes remain elusive. Intestinal microbiota represents an important environmental component implicated in CRC development. Interestingly, microbiota have been shown to modulate DNA methylation in preclinical models but the relationship between tumor-infiltrating bacteria and CIMP status in currently unknown. Our hypothesis is that the gut microbiota affects colonic neoplasia through modulating aberrant DNA methylation in host epigenome. To test this hypothesis, we analyzed CIMP status in CRC patient tumor samples. We used a genome-wide approach to determine the CIMP status by filtering cancer-related sites. A total of 1317 CpG sites were filtered and used to determine distinct CIMP classifications that aligned with well-known characteristics of CIMP cases, including localization in the proximal colon, a higher prevalence in female patients, and a higher frequency of MLH1 hypermethylation. To study the association between CIMP and the gut microbiota, we analyzed the enrichment of four bacterial species associated with CRC, including Bacteroides fragilis, Escherichia coli, Fusobacterium nucleatum, and Klebsiella pneumoniae. Notably, they exhibited higher enrichment in CIMP-Positive tumor samples, except for E. coli. This analysis also identified a group of samples referred to as bacterial "Superhigh," characterized by remarkably high abundances of these three bacterial species. The bacterial Superhigh cases displayed a significant association with CIMP status and MLH1 methylation. We validated the association between the CRC-associated bacteria and CIMP by analyzing the Cancer Genome Atlas (TCGA) 450K methylation array data and whole exome sequencing data. The analysis demonstrated that bacterial Superhigh cases in the TCGA datasets also had significantly higher odds of being CIMP-Positive and having MLH1 methylation. To expand our investigation, we conducted 16S rRNA gene sequencing to identify additional bacterial taxa linked to CIMP. Numerous bacterial genera and species were found to be enriched in CRC tumor tissues, with specific enrichments in CIMP-Positive and CIMP-High groups. Notably, Bergeyella, Campylobacter concisus, and Fusobacterium canifelinum were significantly enriched in CIMP-Positive tumors. Additionally, I studied the causal relationship between gut microbiota and CpG island methylation by colonizing germ-free mice ApcMinΔ850/+;Il10–/– with E. coli NC101 & K. pneumoniae, specific pathogen free bacteria, and the mouse bacterial Consortium. Differential methylation analyses of adjacent normal colon tissue revealed a pronounced tissue side-specific difference, particularly in non-CpG island regions. The tissue specificity diminished with the increasing tumorigenic potential of the microbiota group. Comparisons between microbiota groups and germ-free mice indicated a more significant increase in methylation within CpG islands when gut microbiota with higher tumorigenic potential was present. In conclusion, our study underscores the association between CIMP in CRC and the gut microbiota and the causal relationship between the cut microbiota and CpG island methylation. It highlights specific bacterial taxa that may impact DNA methylation especially in CpG islands and contribute to the development ang progression of CIMP in colorectal cancer. / Biomedical Sciences

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