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Intra-tumoral heterogeneity in NRAS-driven metastatic melanoma

Periodic Reporting for period 1 - INHuMAN (Intra-tumoral heterogeneity in NRAS-driven metastatic melanoma)

Reporting period: 2019-04-01 to 2021-03-31

Despite recent therapeutic advances in cancer treatment, metastasis remains the principal cause of cancer death. An incomplete view of the mechanisms that drive metastasis has been a major barrier to rational development of effective therapeutics and prognostic diagnostics for patients. The mechanisms contributing to metastatic dissemination can be diverse and tumor-type dependent. Skin cancers are prime examples of this. Whereas skin basal cell carcinoma rarely metastasize, cutaneous melanoma are often highly metastatic. Intra-tumor heterogeneity (ITH) has been proposed as a major driver of metastatic dissemination where the magnitude of heterogeneity has not been fully addressed yet. In the action “INHuMAN” we proposed to address the magnitude of intratumoral heterogeneity in melanoma and identify cell populations at the origin of metastasis. Combining single cell RNA sequencing with clinically relevant mouse models we were able to identify and characterize a cell state that presumably sits at the origin of metastasis. Importantly, the data strongly suggested that this population is not fueling tumor growth. Our initial screening assays identified a melanoma population with stemness characteristics indicating that these cells maintain melanoma growth. We developed models and tools to further study this population and by following the fate of individual cells and their spatial distribution in the tissue we identified a specialized cellular niche, called perivascular niche, that maintains tumor growth and is not linked to metastasis. Instead, the metastatic population was driven by a Transcription Factor called PRRX1, where these Metastatic Initiating Cells (MICs) where preferentially at the invading front of primary melanoma tumors. Importantly, the existence and the spatial location of the two cell states were confirmed in human biopsies.
In this call we have characterized this cellular plasticity and based on this data we have initiated projects to better characterize when and how cells transit, in order to block this phenotypic switch. The outcome of this fundamental action can be subjected to translational research that potentially can lead to the development of treatment strategies (personalized or not) to eradicate this devastating disease and improve patient’s survival.
The main goal of the “INHuMAN’’ action was the exhaustive portraying of the diversity and plasticity of melanoma cell states. The call comprised three objectives and two working packages per objective.
In the objective 1 we were able to demonstrated that the transcriptomic landscape of both mouse and human melanoma is richer than previously reported and it can be fully recapitulated in lesions arising in NRAS-mutant mice. Cross-species analyses identified novel and evolutionarily conserved melanoma states, including a highly de-differentiated melanoma stem cell-like state (MSCs) raising the possibility that this population may be sitting at the top of a cellular hierarchy established in these melanoma lesions. Interestingly, another state of interest conserved in human and mouse tumors exhibited mesenchymal-like (EMT) features. Unsupervised in silico analyses predicted the Prrx1 Transcription Factor (TF) as a putative driver of this state.
In turn, the objective 2 was focused on the clinical characterization of the two particular cell states that we identified in our single cell RNA sequencing experiments. The clinical relevance was validated at single cell resolution using scRNA in human biopsies and by multiple imaging approaches in Patient Derived Xenografts and human melanoma biopsies. The spatial location was also assessed where it was found that melanoma stem cells are localized in close proximity to blood vessels while the Metastatic Initiating Cells in the invasive front of primary tumors.
In the third objective first we characterized the MSC population demonstrating that growth of melanoma is hierarchically organized suggesting that these cells are sitting at the apex of this hierarchy. These cells are located in perivascular niches and are able to acquire stemness features fueling growth. In the second WP we characterized the functional relevance and contribution of Metastatic Initiating Cells driven by the transcription factor PRRX1. By performing in vitro and in vivo assays provide direct evidence that Prrx1 marks a population of cells at the origin of metastases.
The outcome of this action is currently under consideration for future publication (Karras et al., under revision, Nature). During this call, I have been able to co author in projects related to melanoma heterogeneity and metastasis that are still ongoing or have been published. Internal biweekly meetings and meetings with collaborators were taking place frequently to discuss the existing progress of the project or future steps. I have been able to attend conferences/meetings and disseminate my work by giving oral talks or poster presentations. Any potential intellectual property that will arise from this action will be determined by Technology Transfer Office of VIB.
The treatment of advanced cancers remains a major clinical challenge. Most recent therapies are limited by high toxicity, and the rapid development of resistance, which is often by different subpopulations that harbour extensive cellular plasticity. Thus, a major future therapeutic challenge is the development of personalized treatment strategies to predict and finally prevent cell plasticity in patients. During this action we addressed the level of intratumor heterogeneity and extensively characterized two main cell states that contribute to growth and metastasis, respectively. In melanoma, the existence of Cancer Stem Cells (CSCs) remains controversial due to the lack of appropriate model systems that allow to track cellular dynamics. Our data indicate that the processes of growth and metastasis are uncoupled and tumors are spatially organized. However, the data do not exclude the possibility that these two populations can switch from one state to the other via exposure to specific extrinsic cues, such as signals emanating from the endothelium.
The study is ongoing where our main focus is on the better characterization of the niches that the cells reside. A series of experimental strategies has been designed by introducing new models and approaches. Part of this strategies is to better characterize the interplay of different melanoma cell states with the microenvironment.
The outcome of this action revealed that the cellular plasticity of melanoma tumors can have major socio-economic impact. Efforts to block phenotypic transitions by developing transcriptional and epigenetic approaches (e.g. chromatin remodeling inhibitors) is likely to be a particularly beneficial therapeutic approach to limiting plasticity and eventually the growth and metastasis of cells. Our data provide the first exhaustive overview of melanoma heterogeneity providing a platform for identification of druggable targets for more effective therapeutic approaches that intercept the disease before its lethal spreading to vital organs. Importantly, converging lines of evidence from our experimental mouse studies and clinical samples can unravel vulnerabilities of the Melanoma Stem Cells and the Metastatic Initiating Cells that can lead to effective target and eradication.
MSCA summary schematic