An in vivo avian model of human melanoma to perform rapid and robust preclinical studies.

Metastatic melanoma patients carrying a BRAFV600 mutation can be treated with a combination of BRAF and MEK inhibitors (BRAFi/MEKi), but innate and acquired resistance invariably occurs. Predicting patient response to targeted therapies is crucial to guide clinical decision. We describe here the development of a highly efficient patient-derived xenograft model adapted to patient melanoma biopsies, using the avian embryo as a host (AVI-PDXTM ). In this in vivo paradigm, we depict a fast and reproducible tumor engraftment of patient samples within the embryonic skin, preserving key molecular and phenotypic features. We show that sensitivity and resistance to BRAFi/MEKi can be reliably modeled in these AVI-PDXTM , as well as synergies with other drugs. We further provide proof-of-concept that the AVI-PDXTM models the diversity of responses of melanoma patients to BRAFi/MEKi, within days, hence positioning it as a valuable tool for the design of personalized medicine assays and for the evaluation of novel combination strategies.

Environmental cues from neural crest derivatives act as metastatic triggers in an embryonic neuroblastoma model.

Embryonic malignant transformation is concomitant to organogenesis, often affecting multipotent and migratory progenitors. While lineage relationships between malignant cells and their physiological counterparts are extensively investigated, the contribution of exogenous embryonic signals is not fully known. Neuroblastoma (NB) is a childhood malignancy of the peripheral nervous system arising from the embryonic trunk neural crest (NC) and characterized by heterogeneous and interconvertible tumor cell identities. Here, using experimental models mimicking the embryonic context coupled to proteomic and transcriptomic analyses, we show that signals released by embryonic sympathetic ganglia, including Olfactomedin-1, induce NB cells to shift from a noradrenergic to mesenchymal identity, and to activate a gene program promoting NB metastatic onset and dissemination. From this gene program, we extract a core signature specifically shared by metastatic cancers with NC origin. This reveals non-cell autonomous embryonic contributions regulating the plasticity of NB identities and setting pro-dissemination gene programs common to NC-derived cancers.

An avian embryo patient-derived xenograft model for preclinical studies of human breast cancers.

Lack of preclinical patient-derived xenograft cancer models in which to conduct large-scale molecular studies seriously impairs the development of effective personalized therapies. We report here an in vivo concept consisting of implanting human tumor cells in targeted tissues of an avian embryo, delivering therapeutics, evaluating their efficacy by measuring tumors using light sheet confocal microscopy, and conducting large-scale RNA-seq analysis to characterize therapeutic-induced changes in gene expression. The model was established to recapitulate triple-negative breast cancer (TNBC) and validated using TNBC standards of care and an investigational therapeutic agent.

Blocking SHH/Patched Interaction Triggers Tumor Growth Inhibition through Patched-Induced Apoptosis.

The Sonic Hedgehog (SHH) pathway plays a key role in cancer. Alterations of SHH canonical signaling, causally linked to tumor progression, have become rational targets for cancer therapy. However, Smoothened (SMO) inhibitors have failed to show clinical benefit in patients with cancers displaying SHH autocrine/paracrine expression. We reported earlier that the SHH receptor Patched (PTCH) is a dependence receptor that triggers apoptosis in the absence of SHH through a pathway that differs from the canonical one, thus generating a state of dependence on SHH for survival. Here, we propose a dual function for SHH: its binding to PTCH not only activates the SHH canonical pathway but also blocks PTCH-induced apoptosis. Eighty percent, 64%, and 8% of human colon, pancreatic, and lung cancer cells, respectively, overexpressed SHH at transcriptional and protein levels. In addition, SHH-overexpressing cells expressed all the effectors of the PTCH-induced apoptotic pathway. Although the canonical pathway remained unchanged, autocrine SHH interference in colon, pancreatic, and lung cell lines triggered cell death through PTCH proapoptotic signaling. In vivo, SHH interference in colon cancer cell lines decreased primary tumor growth and metastasis. Therefore, the antitumor effect associated to SHH deprivation, usually thought to be a consequence of the inactivation of the canonical SHH pathway, is, at least in part, because of the engagement of PTCH proapoptotic activity. Together, these data strongly suggest that therapeutic strategies based on the disruption of SHH/PTCH interaction in SHH-overexpressing cancers should be explored. SIGNIFICANCE: Sonic Hedgehog-overexpressing tumors express PTCH-induced cell death effectors, suggesting that this death signaling could be activated as an antitumor strategy.

The dependence receptor TrkC regulates the number of sensory neurons during DRG development.

The development of the sensory nervous system is the result of fine-tuned waves of neurogenesis and apoptosis which control the appropriate number of precursors and newly generated neurons and orient them toward a specific lineage. Neurotrophins and their tyrosine-kinase receptors (RTK) orchestrate this process. They have long been in the scope of the neurotrophic theory which established that a neuron is committed to die unless a trophic factor generated by its target provides it with a survival signal. The neural death has thus always been described as a "default" program, survival being the major player to control the number of cells. New insights have been brought by the gain of function studies which recently demonstrated that TrkC (NTRK3) is a "dependence receptor" able to actively trigger apoptosis in absence of its ligand NT-3. In order to address the role of TrkC pro-apoptotic activity in the control of sensory neurons number, we generated a TrkC gene-trap mutant mice. We found out that this new murine model recapitulates the sensory phenotype of TrkC constitutive mutants, with reduced DRG size and reduced number of DRG neurons. We engineered these mice strain with a lacZ reporter in order to follow the fate of neurons committed to a TrkC lineage and observed that they are specifically protected from NT-3 mediated apoptosis in NT-3/TrkC double knock-out embryos. Finally, using a chicken model we demonstrated that silencing NT-3 emanating from the ventral neural tube induced apoptosis in the DRG anlage. This apoptosis was inhibited by silencing TrkC. This work thus demonstrates that, during in vivo DRG development, TrkC behaves as a two-sided receptor transducing positive signals of neuronal survival in response to NT-3, but actively inducing neuronal cell death when unbound. This functional duality sets adequate number of neurons committed to a TrkC identity in the forming DRG.

Hey1- and p53-dependent TrkC proapoptotic activity controls neuroblastoma growth.

The neurotrophin-3 (NT-3) receptor tropomyosin receptor kinase C (TrkC/NTRK3) has been described as a dependence receptor and, as such, triggers apoptosis in the absence of its ligand NT-3. This proapoptotic activity has been proposed to confer a tumor suppressor activity to this classic tyrosine kinase receptor (RTK). By investigating interacting partners that might facilitate TrkC-induced cell death, we have identified the basic helix-loop-helix (bHLH) transcription factor Hey1 and importin-α3 (karyopherin alpha 4 [KPNA4]) as direct interactors of TrkC intracellular domain, and we show that Hey1 is required for TrkC-induced apoptosis. We propose here that the cleaved proapoptotic portion of TrkC intracellular domain (called TrkC killer-fragment [TrkC-KF]) is translocated to the nucleus by importins and interacts there with Hey1. We also demonstrate that Hey1 and TrkC-KF transcriptionally silence mouse double minute 2 homolog (MDM2), thus contributing to p53 stabilization. p53 transcriptionally regulates the expression of TrkC-KF cytoplasmic and mitochondrial interactors cofactor of breast cancer 1 (COBRA1) and B cell lymphoma 2-associated X (BAX), which will subsequently trigger the intrinsic pathway of apoptosis. Of interest, TrkC was proposed to constrain tumor progression in neuroblastoma (NB), and we demonstrate in an avian model that TrkC tumor suppressor activity requires Hey1 and p53.