Generating in vitro models of NTRK-fusion mesenchymal neoplasia as tools for investigating kinase oncogenic activation and response to targeted therapy.

The discovery of neurotrophic tyrosine receptor kinase (NTRK) gene fusions as pan-tumor oncogenic drivers has led to new personalized therapies in oncology. Recent studies investigating NTRK fusions among mesenchymal neoplasms have identified several emerging soft tissue tumor entities displaying various phenotypes and clinical behaviors. Among them, tumors resembling lipofibromatosis or malignant peripheral nerve sheath tumors often harbor intra-chromosomal NTRK1 rearrangements, while most infantile fibrosarcomas are characterized by canonical ETV6::NTRK3 fusions. However, appropriate cellular models to investigate mechanisms of how kinase oncogenic activation through gene fusions drives such a wide spectrum of morphology and malignancy are lacking. Progress in genome editing has facilitated the efficient generation of chromosomal translocations in isogenic cell lines. In this study we employ various strategies to model NTRK fusions, including LMNA::NTRK1 (interstitial deletion) and ETV6::NTRK3 (reciprocal translocation) in human embryonic stem (hES) cells and mesenchymal progenitors (hES-MP). Here, we undertake various methods to model non-reciprocal, intrachromosomal deletions/translocations by induction of DNA double strand breaks (DSBs) exploiting either the repair mechanisms of homology directed repair (HDR) or non-homologous end joining (NHEJ). Expression of LMNA::NTRK1 or ETV6::NTRK3 fusions in either hES cells or hES-MP did not affect cell proliferation. However, the level of mRNA expression of the fusion transcripts was significantly upregulated in hES-MP, and phosphorylation of the LMNA::NTRK1 fusion oncoprotein was noted only in hES-MP but not in hES cells. Similarly, an NTRK1-driven transcriptional profile related to neuronal and neuroectodermal lineage was upregulated mainly in hES-MP, supporting the importance of appropriate cellular context in modeling cancer relevant aberrations. As proof of concept of the validity of our in vitro models, phosphorylation was depleted by two TRK inhibitors, Entrectinib and Larotrectinib, currently used as targeted therapy for tumors with NTRK fusions.

Generation of human embryonic stem cell models to exploit the EWSR1-CREB fusion promiscuity as a common pathway of transformation in human tumors.

Chromosomal translocations constitute driver mutations in solid tumors and leukemias. The mechanisms of how related or even identical gene fusions drive the pathogenesis of various tumor types remain elusive. One remarkable example is the presence of EWSR1 fusions with CREB1 and ATF1, members of the CREB family of transcription factors, in a variety of sarcomas, carcinomas and mesotheliomas. To address this, we have developed in vitro models of oncogenic fusions, in particular, EWSR1-CREB1 and EWSR1-ATF1, in human embryonic stem (hES) cells, which are capable of multipotent differentiation, using CRISPR-Cas9 technology and HDR together with conditional fusion gene expression that allows investigation into the early steps of cellular transformation. We show that expression of EWSR1-CREB1/ATF1 fusion in hES cells recapitulates the core gene signatures, respectively, of angiomatoid fibrous histiocytoma (AFH) and gastrointestinal clear cell sarcoma (GI-CCS), although both fusions lead to cell lethality. Conversely, expression of the fusions in hES cells differentiated to mesenchymal progenitors is compatible with prolonged viability while maintaining the core gene signatures. Moreover, in the context of a mesenchymal lineage, the proliferation of cells expressing the EWSR1-CREB1 fusion is further extended by deletion of the tumor suppressor TP53. We expect the generation of isogenic lines carrying oncogenic fusions in various cell lineages to expand our general understanding of how those single genetic events drive tumorigenesis while providing valuable resources for drug discovery.