Cutaneous lymphomas represent a heterogeneous group of rare diseases that need access to referral center for diagnosis and treatment. The aggressive cutaneous lymphomas, including transformed mycosis fungoides (T-MF), Sézary syndrome (SS) and primary cutaneous large B-cell lymphoma – leg type (PCLBCL-LT), occur, for most of them, in elderly patients and represent unmet needs for tailored therapy. We aimed at characterizing genetic alterations and signaling pathways to establish new diagnostic, prognostic or predictive biomarkers. In cutaneous T-cell lymphomas (CTCL), we identified the high prevalence of some genetic alterations associated with subtypes and progression (DUSP22 and TP53 genes) and the role of telomerase activity in CTCL oncogenesis and progression. To understand the functions of such alterations, we established new cell lines and original preclinical models of CTCL displaying many features of the disease such as epidermotropism, blood spreading and dissemination to organs. The maturation/differentiation stage of CTCL is much debated according their phenotype (naïve, central memory or effector memory T-cells). To determine whether CTCL arise from different T-cell stages, exhibit aberrant phenotypes challenging such a characterization, or may exhibit intra-clonal heterogeneity or plasticity either at spatial or tissue level or over time and progression of the disease, we are developing whole exome sequencing, RNAseq and scRNAseq on our original CTCL models. Moreover, our team has recently joined the Euroflow network aiming at standardizing SS characterization by flow cytometry.

In cutaneous large B-cell lymphomas, we identified MYD88 and BCR mutations as respectively diagnostic and predictive biomarkers of treatment response to rituximab and polychemotherapy (R-PCT). Given that MYD88 mutation lead to activation of NF-kB pathway, we conducted the only clinical trial to date in these lymphomas with lenalidomide. Using RNA sequencing combined with proteomic analysis on the patient samples we currently identify the altered genomic and signaling pathways, actionable targets and predictive biomarkers in both cutaneous large B-cell lymphomas and in skin carcinomas at different stages of carcinogenesis. Functional validation of therapeutic targets as well as evaluation of response to conventional or innovative drugs are under investigation.

Concerning cutaneous squamous cell carcinoma (cSCC), we are creating a biobank of the following human samples: actinic keratosis (AK), in situ cSCC, infiltrative cSCC and, recurrent/metastatic cSCC (reference CPP: AU 1485; Ref ID-CRB: 2018-A03096-49; involved biobank: “Centre de Ressources Biologiques” (CRB)). Taking advantage of our expertise in DNA and RNA sequencing, we will examine the genetic landscape of human cSCC at different stages of carcinogenesis.

Specific aim-1: Understanding oncogenesis to recognize new diagnostic, and predictive biomarkers and for identification and validation of therapeutic targets

Specific aim-2: Characterization of genetic features of squamous cell carcinomas

Owing to its central function in numerous cellular processes including cell cycle progression, apoptosis/senescence and autophagy, energy metabolism has a crucial role in carcinogenesis. It is now widely accepted that mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis cooperate to sustain the variable energy demands and anabolic needs of cancer cells and that cancer cells achieve those needs through various mechanisms. The critical role of mitochondria in carcinogenesis and gene regulation was further evidenced by recent studies on various oncometabolites produced by the TCA cycle and by the role of glutaminolysis. A better understanding of what determines tumor bioenergetics is also crucial for developing adapted metabolic therapies, as proposed for isocitrate dehydrogenase (IDH) 1 and 2 mutant tumors.

Focusing on the role of oxidative and energy metabolism in UVB-induced carcinogenesis, we have recently found that early changes in oxidative and energy metabolism play a critical role in initiation of UVB-induced cSCC (PMID: 29925003, 30878676, 31551419). Our results indicated that specific metabolic modifications occur at the very early stage of photocarcinogenesis. Deciphering the molecular mechanisms underlying those early metabolic changes, we have shown that a specific mode of electrons fueling to the electron transport chain (ETC) occurs following chronic irradiation. Indeed, the distal part of the ETC is upregulated in precancerous lesions owing to the over-activation of dihydroorotate dehydrogenase (DHODH), the enzyme catalyzing the fourth step of pyrimidine synthesis. Mice with decreased DHODH activity or impaired ETC in keratinocytes failed to develop pre-malignant and malignant lesions (PMID: 29925003). Our results also showed that DHODH is a promising target for chemoprevention and combination therapy of UVB-induced cSCCs (PMID: 31551419). Although bioenergetic vulnerabilities-based therapies of human cancers have been recently received great attention, several studies highlighted interpatient metabolic heterogeneities and thus the importance of metabolic phenotyping in cancer therapy. Given that little attention has been given to the bioenergetic profiling of cutaneous carcinomas and lymphomas, our main objective is to characterize their metabolic features.

Specific aim-1: Whether targeting energy metabolism vulnerabilities (e.g. de novo pyrimidine and/or purine biosynthesis pathways and OXPHOS) could be exploited as new preventive strategies and/or promising curative therapeutic options.

Specific aim-2: Whether metabolic features could be used as prognostic and diagnostic biomarkers to identify precancerous and cancerous lesions at high evolution risk.

Specific aim-3: Identifying metabolic features of cutaneous T-cell lymphoma

Increasing evidence highlights the contribution of the skin microenvironment components to skin physiology in general and to skin tumor development in particular.

In addition to its direct mutagenic effects, solar UV irradiation also directly affects the skin microenvironment by influencing the interplay between keratinocytes, fibroblasts, melanocytes, immune cells and endothelial cells. The collaboration between different components of the skin establishes a foundation for an appropriate and coordinated response to deleterious effect of UV radiation. This network response to UV exposure provides a photoprotective barrier through affecting constitutive and UV-induced pigmentation, stratum corneum thickness, metabolic features and immune and angiogenic properties of skin (PMID: 28990413, PMID: 29938913). Although the skin responses to UV radiation have been studied at the individual cell type level, little attention has been given to the mentioned network response. Taking advantage of our relation with the national reference center for rare skin diseases at Bordeaux Hospital University, we studied some of UV-related pigmentary disorders using 3D skin equivalents and developed the models recapitulating senile lentigo (PMID: 24533682), melasma (PMID: 30478899), and xeroderma pigmentosum (PMID: 21123941). Using these models, we have highlighted the impact of dermal components on skin pigmentary profile (PMID: 31692218, PMID: 31954725, PMID: 32633087). Furthermore, our recent studies highlighted that sensitivity of cutaneous infantile hemangioma (IH) to propranolol (the first-line therapy for severe IH) rely on a cross talk between lesional vascular cells and stromal telocytes, which are dendritic cells that form a distinctive peripheral layer in IH vascular structures (PMID: 33558238, https://doi.org/10.3390/ijms23095140). Therefore, one of the our objectives is to unravel the relationship between keratinocytes, fibroblasts, melanocytes, telocytes, immune cells and endothelial cells during skin response to UV exposure, with particular attention to the contribution of pigmentation and angiogenesis in photoprotection and UV-induced carcinogenesis.

The relationship between different components of skin microenvironment and tumor cells now represents one of the most important network to elucidate. Therefore, our other objective is to study the role of microenvironment is skin cancers. The microenvironment landscape of skin carcinomas and cutaneous lymphomas according to the stage of the tumors is revealed using scRNAseq, spatial transcriptomics and our in house multiplex staining strategies. To decipher the relationships between the tumor cells and the microenvironment, we use classic autologous 2D co-cultures including tumor cells, epidermal cells and various dermal components (extracellular matrix components, fibroblasts, telocytes, immune and endothelial cells). Functional relevance of the identified crosstalks between different components of tumor microenvironment and tumor cells are then studied using innovative in vitro 3D models including cellular capsule technology (PMID: 34555842), skin equivalent and in vivo mouse models (xenograft models, xenopus transgenic mice, and skin humanized mice). To verify the role of factors involved in these crosstalks, they are overexpressed or silenced (shRNA or CRISP/CAS9) using lentiviral vectors and studied in our models.

This will also be studied in vivo by transplanting the patient tumor with its own environment in CAM and NSG mouse models. These preclinical models (3D culture models together with cells lines and PDX) will allow to validate at the functional level the molecular pathways involved in drug resistance, the role of tumor environment and to validate putative therapeutic targets.

Specific aim-1: Skin microenvironment in skin physiology

Specific aim-2: Skin microenvironment in photoprotection and

Specific aim-3: Functional characterization of the cutaneous lymphoma microenvironment