Oncogenic Competence at the Cellular and Multicellular Level in Melanoma

Oncogenes cause malignant transformation only in certain cellular contexts, a phenomenon called oncogenic competence. In melanoma, tumour initiation upon oncogene expression activates a neural crest lineage program. My work has shed some light on why a neural crest-like state is required and particularly susceptible to oncogenic transformation and shown that an epigenetic developmental program regulates such a state. My lab will take a multidisciplinary approach using my expertise in mouse biology, cellular engineering, and human pluripotent stem cell (hPSC)-based technologies to study the epigenetic regulation of oncogenic competence during melanoma initiation and progression. Finally, we will define how oncogenic competence is regulated at the cell-intrinsic and multicellular levels depending on the microenvironment. Oncogenic competent progenitor cells express a distinct profile of epigenetic-related factors. Among those, ATAD2, an ATPase- and bromodomain-containing protein, regulates oncogenic competence in melanoma, activates a developmental signature typical of the neural crest and regulates the MAPK pathway. ATAD2 is necessary and sufficient for tumor initiation and confers invasive properties to hPSC-derived melanoma cells. Furthermore, ATAD2 induces the expression of many epigenetic factors known to play a role in melanoma, and many of those factors are direct targets. My lab will investigate the developmental chromatin programs and the role of ATAD2 in the epigenetic regulation of both melanoma initiation and progression.A progenitor cell-related signature is often related to melanoma invasion to distant organs, and the brain is one of the most frequent sites affected by metastatic melanoma cells. My lab will use a cutting-edge hPSC-based brain organoid model, that I have developed, to study oncogenic competence in the human brain microenvironment, the cellular cross-talk between melanoma cells and the brain cells, cancer cell adaptation and niche remodelling. The brain microenvironment imposes a distinct selective pressure on the cancer cell. The 3 cell types – neurons, astrocytes, and microglia – build the neuroinflammatory axis, which involves complex cellular cross-talk that promotes homeostasis and neuronal function during health but can play a pro-metastatic role in the context of brain metastases. Challenging access to brain cells, especially in the human context, and difficulties maintaining those cells in culture, have hampered the study of melanoma cells in the human brain microenvironment. Studies using genetically engineered mouse models have been proven to be very efficacious and instructive. However, some aspects of the neuroinflammatory axis are unique to humans reflecting the species-specific differences in the cell types of the brain. Using both melanoma mouse models and the hPSC-derived brain organoid platform, we will investigate cellular cross-talk and melanoma cell adaption to the brain microenvironment and highlight the mechanisms responsible for the instruction and the remodeling of the brain niche.Aim A) Investigation of the developmental chromatin programs controlling oncogenic competence during melanoma initiation and progression. We will investigate the epigenetic regulatory landscape and the role of ATAD2 in the epigenetic regulation of oncogenic competence during melanoma initiation and progression. We will define the upstream mechanisms that regulate the expression of ATAD2 and the acquisition of an oncogenic competent state and, finally, build a melanoma mouse model to study the role of Atad2 in vivo.-A.1: Defining the epigenetic landscape regulating a progenitor signature in hPSC-derived melanoma cells (PhD student #1): inducible KO of developmental chromatin factors (ATAD2, BPTF, EZH2) using the dox-inducible Cas9 system, Cut&Run, RNA-seq and ATAC-seq analyses in both WT hPSC-derived progenitor cells and hPSC-derived melanoma cells. coIP of ATAD2 followed by mass spectrometry analysis to identify further co-factors. Overexpression compensatory studies.-A.2: Targeting developmental chromatin programs and oncogenic competence to define new therapeutic approaches (Postdoctoral fellow #1): inducible KO of developmental chromatin factors (25 factors) to induce differentiation and loss of a progenitor signature in drug-resistant melanoma cells and hPSC-derived melanoma cells. Follow-up studies of the candidates with Cut&Run, RNA-seq and ATAC-seq. Generation of an ATAD2-GFP+ hPSC line. Screening with a 3038 FDA-approved drugs library to define the upstream regulator of ATAD2 expression. Validation of the hits and their application on drug-resistant melanoma cells, including an ATAD2 inhibitor (GSK8814).-A.3: Generation of the Atad2lox/lox melanoma mouse model (Postdoctoral fellow #1): Expansion of the Atad2lox/lox mouse line and crossing with the Tyr::CreER BrafCA/+ Ptenflox/flox line. KO validation, study of the role of Atad2 in vivo (tumor formation vs progression).Aim B) Study of oncogenic competence at the multicellular level in the brain. We will identify the mechanisms that regulate melanoma cell adaptation to the brain microenvironment and niche remodeling. We will investigate the role of developmental chromatin programs in the formation of brain metastasis, and how the niche might select and/or regulate those programs. To this aim, that lab will use both the hPSC-derived brain organoid platform and mouse models of melanoma brain metastasis.-B.1: Investigation of the cellular cross-talk (PhD Student #2): hPSC-derived brain organoids analyzed at 4 different time points post exposure to human melanoma cells. The features of cellular cross-talk will be analyzed by TUNEL, RT-qPCR, IF, ELISA, EM, microelectrode array. Finally, to proof neuron-melanoma cross-talk we will use rabies virus (EnvA G-Deleted Rabies- H2B-mCherry and LV-pBOB-synP-HTB). At each time point, we will quantify the expression of key inflammatory and anti-inflammatory markers (M1/M2 genes, A1/A2 genes). The release of complement C3, an inflammatory cytokine secreted by both astrocytes and microglia, will be measured by ELISA.-B.2: Investigation of melanoma cell adaptation to the brain microenvironment (Postdoctoral fellow #2): scRNA-seq of hPSC-derived brain organoids exposed to 2 melanoma cells lines (ATAD2 high and low expression levels) at 4 different time points: t0 (baseline, brain organoid without melanoma cells), t48h (48 hours post organoid + melanoma cells), t7days (7 days post organoid + melanoma cells), t14days (14 days post organoid + melanoma cells). Genetic perturbation studies of driver candidates, including ATAD2, and transplantation studies in NSG mice. Generation of brain tropic B6 mouse melanoma cell lines, validation studies in immunocompetent melanoma mice and in melanoma patient samples (collaboration with Prof. Levesque and Prof. Dummer at the USZ, see “Other annexes”).-B.3: Study of the mechanisms responsible for the instruction and the remodeling of the niche (PhD student #2): Analysis of the scRNA-seq (B.2) for the transcriptional changes occurring in the brain niche. Genetic perturbation studies in the brain-specific cell types using the dox-inducible Cas9 hPSC line to induce and anti-metastatic brain niche. Follow-up studies with Cut&Run and RNA-seq. Validation studies of the brain niche-specific changes using both melanoma brain metastasis mouse model (B.2) and patient-derived samples.Our work will provide mechanistic insights into the process of oncogenic competence and its regulation both at the cell-intrinsic and extrinsic levels, depending on the microenvironment. We will investigate the role played by developmental chromatin factors in the regulation of oncogenic competence during melanoma initiation and metastasis formation. Furthermore, we will define novel approaches to target oncogenic competence, exploit susceptibilities of melanoma brain metastasis and promote an anti-metastatic niche.