University of Kentucky Center for Cancer and Metabolism (Project 2)

The two major pathobiologies of non-small cell lung cancer, adenocarcinoma (ADC) and squamous cell carcinoma (SCC), are historically treated as separate diseases. However, mounting evidence shows that epigenetic reprogramming from ADC to a more SCC fate allows lung cancers to evade therapies. My team recently developed an isogenic lung cancer model in which lineage switching from ADC to SCC was epigenetically controlled through loss of Polycomb Repressive Complex 2 (PRC2) gene repression of squamous genes. The lineage switch was reliant upon deletion of Lkb1, but not every Lkb1-null tumor reprogrammed to the squamous fate. Previous steady state metabolism showed that the methyl donor for PRC2, S-adenosyl methionine (SAM) is decreased significantly and cystathionine is increased ignificantly in SCC cells relative to ADC cells. These data suggest that SAM pools are limited in SCC due to increased catalysis of homocysteine to cystathionine by the enzyme cystathionine beta-synthase (CBS). My central hypothesis is that increased CBS activity drives epigenetic reprogramming of lung ADC to an aggressive and therapy-resistant state through reduction of SAM pools and destabilization of PRC2, leading to expression of squamous genes. In Aim 1, I will use both human two-dimensional cultures and murine three-dimensional organoid cultures and perform stable isotope-resolved metabolomics to trace homocysteine utilization in ADC and SCC tumors. I will use genetic and pharmacological approaches to modulate CBS activity, and link changes in homocysteine metabolism to PRC2 stability and activity using a variety of molecular biology techniques. I will also trace directly the incorporation of SAM onto H3K27me3 in ADC and SCC cells in cells with modulated CBS expression and activity. In Aim 2, I will use the innovative isogenic lineage switching model and liquid diet to administer a methionine tracer in vivo. I will again trace homocysteine usage in tumors that are genetically modulated to have decreased CBS activity. I expect that altered CBS activity and homocysteine metabolism will influence lung cancer epigenetic fate as measured by histological and biochemical methods including ChIP-seq and RNA-seq. Comparison to the in vitro results will allow for dissection of the contribution of the tumor microenvironment to cellular plasticity. Lastly, I will use human lung cancer patient samples and data to confirm that CBS and PRC2 activities influence epigenetic fate of primary lung cancer cells and correlate these changes to therapeutic outcomes. Expected outcomes will establish how homocysteine usage orchestrates epigenetic fate, and demonstrate the role of CBS, an understudied enzyme in lung cancer, in determining lung cancer lineage plasticity. This research is important for understanding how to direct epigenetic plasticity of lung cancers to improve treatment outcomes.