A novel FLI1+ hybrid cell state in melanoma residual disease

Abstract

Melanoma, formed from the malignant transformation of the pigment producing melanocytes, is the most aggressive form of skin cancer. In the UK, melanoma is the 5th most common cancer and its incidence has more than doubled since the early 1990s. Despite recent advances in treatment options, patients with metastatic melanoma typically have a poor prognosis, due to low response rate to immune therapies (e.g., checkpoint inhibitors) and high frequency of resistance to targeted therapies (e.g., BRAF inhibitors). Therefore, it is a necessity to develop more effective and durable therapeutic options for patients with metastatic melanoma. Melanoma is comprised of diverse cell populations of highly plastic transcriptional cell states which are considered key drivers of therapy resistance and disease progression. Previously, we showed via fate mapping that the cells which survive during disease regression, known as residual disease, directly contribute to tumour relapse (Travnickova et al., 2022). Thus, emphasising the importance of identifying and functionally interrogating the transcriptional cell states that arise and persist during disease regression and recurrence. To interrogate this heterogeneity and plasticity in melanoma, I have used an adult zebrafish model of cutaneous melanoma, in which I can follow tumour regression and recurrence, just as we see in patients as a result of therapy resistance. The origin of my project came from single cell RNA-sequencing of zebrafish melanomas completed by Travnickova et al., (2019), which showed a tumour subpopulation in residual disease unexpectedly expressing fli1. FLI1, an ETS transcription factor, is a key transcriptional regulator in development and homeostasis, and is predominately expressed in hematopoietic and endothelial cells. However, the functional significance of aberrant FLI1 expression in melanoma was unknown. To investigate this fli1+ tumour cell state, I first generated a Tg(fli1:GFP, crestin:mCherry) zebrafish line on the mutant melanoma background. Using IHC-IF, I was able to visualise fli1+ tumour cells in primary tumours, in persister cells at the residual disease site and in relapsed melanomas. Importantly, I demonstrated that this cell state is relevant to human disease, as I also detected FLI1+ melanoma cells in both patient biopsies and scRNA-sequencing data. Next, I used the Tg(fli1:GFP, crestin:mCherry) zebrafish melanoma model to quantify the fli1+ melanoma subpopulation in primary and regressed tumours using flow cytometry. Excitingly, these data showed that the fli1+ cell state is specifically enriched in residual disease, relative to the progressing tumour. Importantly, preliminary results from ongoing in vitro experiments generating BRAF inhibitor (Vemurafenib) resistant patient cell lines, indicate that FLI1 is also upregulated in response to prolonged Vemurafenib treatment. This provides an important link to the clinic, suggesting the enrichment observed in our genetic model of residual disease, is also conserved in response to targeted therapy. To further understand the dynamics of the fli1+ cell state, I successfully developed a tamoxifen inducible dual marker lineage tracing strategy, which enables a fluorescent ‘switch’ of the fli1+ melanoma cells, allowing tracking of this cell state through disease stages. Using this system in combination with IHC-IF and RNAscopeTM, to assess fli1 expression in fluorescently ‘switched’ cells, I demonstrated that the fli1+ tumour cells persist beyond residual disease and contribute to tumour relapse. In addition, this strategy revealed the plasticity of the fli1+ cell state, showing that fli1+ melanoma cells in residual disease can alter their transcriptional identity and turn off fli1 expression during tumour relapse. Next, to better understand the cellular identity of the fli1+ cell state, I isolated fli1+ tumour cells from primary and regressed zebrafish melanomas and performed transcriptomic profiling. Crucially, differential gene expression analysis showed that fli1 expression is more than simply a single marker gene, rather representative of a transcriptionally distinct tumour cell state. Furthermore, pathway analysis revealed that the fli1+ tumour cells are hybrid in nature, maintaining melanoma gene expression, while also being enriched for mesenchymal gene signatures. Moreover, a large proportion of the differentially expressed genes of the fli1+ cell state are both targets of Fli1 and expressed in the neural crest lineage during early development, suggesting that Fli1 may be driving a developmental mesenchymal programme, which could prove critical for tumour cell survival during regression. Therefore, to determine whether Fli1 is sufficient to drive the mesenchymal programme in vivo, and assess the impact this has on response to treatment and tumour relapse, I generated an inducible fli1 over-expression transgenic zebrafish line. Early validation experiments in embryos are promising and indicate this line will be effective in driving fli1 over-expression in melanoma tumours in adult zebrafish. Together, this work identifying and characterising this novel fli1+ cell state will better inform our understanding of tumour heterogeneity and plasticity in melanoma residual disease and how to combat therapy resistance