Exploring autophagy as a cellular response to fusion oncogene-induced stress - PROJECT SUMMARY Rare cancers cumulatively account for about 20% of all cancer diagnoses, however, there are fewer available resources, models, and patient samples to enable their study as compared to common cancers. This has created in a significant gap in our understanding of their tumorigenesis. Many of rare cancers harbor specific and recurrent chromosomal translocations, resulting in unique fusion oncogenes which confer aberrant and/or neomorphic function of oncogenes, but the exact role of most fusions in the development of rare cancers remains uncharacterized. To enable high-throughput study of fusion oncogenes, we have constructed the Rare Cancer Fusion Oncogene Library (RCFOL), which is a collection of over 2.5k barcoded open reading frame (ORF) sequences which have been optimized for multiplexed functional genetics studies. In preliminary experiments with the library, I identified that fusions isolated from soft-tissue sarcomas caused fitness deficits in human mammary epithelial cells (HMECs), with EWSR1::CREB1 inducing autophagy-dependent cell death (ADCD) in response to oncogenic stress paired with upregulation of the innate immune response. However, fibroblasts were able to stably express EWSR1::CREB1 with no impact on cellular health, suggesting that certain cell types may be either “permissive”, or “non-permissive” to fusion oncogene expression. Thus, I hypothesize that the induction of autophagy-dependent cell death via innate immune signaling is a conserved response to fusion expression in non-permissive cell-types. In Aim 1, I will characterize the role of IL-6 and differential fusion binding patterns in the selective induction of ADCD in EWSR1::CREB1-expressing HMECs. I will modulate levels of IL-6 using short-hairpin RNAs and monitor autophagic flux, localization of autophagy regulators, and cell death through microscopy, immunocytochemistry, and flow cytometry. To identify if EWSR1::CREB1 directly influences the expression of autophagy and innate-immune response genes, I will conduct multi-CUT&Tag to map fusion binding patterns in both HMECs and BJ fibroblasts. These experiments will reveal how the underlying differences between fusion permissive and non-permissive cells result in disparate phenotypes in response to fusion expression. In Aim 2, I will profile if autophagy is a conserved tumor-suppressive response to oncogenic stress. I will conduct barcoded multiplexed proliferation assays of the fusion ORFs in the RCFOL paired with the GFP-LC3-RFP autophagic flux reporter. To validate which individual fusions induce ADCD and upregulate the innate immune response, I will analyze autophagic flux, cell death, and cytokine secretion. The experiments of Aim 2 will determine if the mechanisms by which fusion expression is restricted to certain cell-types are ubiquitous or highly specific to individual fusions. This proposal will characterize the mechanism of ADCD in EWSR1::CREB1-expressing HMECs, and profile the scope of ADCD as a response to fusion oncogene- mediated stress. While conducting this research, my mentorship team and I will work to improve my expertise in functional genomics and cell biology, while bolstering my mentorship and scientific communication skills.
