Modeling Extraordinary Response Through Targeting Secondary Alterations in Fusion-Associated Sarcoma.

PURPOSE: Targeted therapy in translocation-associated sarcomas has been limited to oncogenic activation of tyrosine kinases or ligands while gene fusions resulting in aberrant expression of transcription factors have been notoriously difficult to target. Moreover, secondary genetic alterations in sarcomas driven by translocations are uncommon, comprising mostly alterations in tumor suppressor genes (TP53, CDKN2A/B). Our study was triggered by an index patient showing a dramatic clinical response by targeting the secondary BRAF V600E mutation in a metastatic angiomatoid fibrous histiocytoma (AFH) harboring the typical EWSR1::CREB1 fusion. MATERIALS AND METHODS: The patient, a 28-year-old female, was diagnosed with an AFH of the thigh and followed a highly aggressive clinical course, with rapid multifocal local recurrence within a year and widespread distant metastases (adrenal, bone, liver, lung). The tumor showed characteristic morphologic features, with histiocytoid cells intermixed with hemorrhagic cystic spaces and lymphoid aggregates. In addition to the pathognomonic EWSR1::CREB1 fusion, targeted DNA sequencing revealed in both primary and adrenal metastatic sites a hot spot BRAF V600E mutation and a CDKN2A/B deletion. Accordingly, the patient was treated with a BRAF-MEK inhibitor combination (encorafenib/binimetinib) showing an excellent but short-lived response. RESULTS: Using a CRISPR-Cas9 approach, we introduced the BRAF c.1799 T>A point mutation in human embryonic stem (hES) cells harboring a conditional EWSR1 (exon7)::CREB1 (exon7) translocation and further differentiated to mesenchymal progenitors (hES-MP) before fusion expression. The cells maintained the fusion transcript expression and the AFH core gene signature while responding to treatment with encorafenib and binimetinib. CONCLUSION: These results highlight that additional targeted DNA NGS in chemotherapy-resistant translocation-associated sarcomas may reveal actionable oncogenic drivers occurring as secondary genetic events during disease progression.

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.

PRC2-Inactivating Mutations in Cancer Enhance Cytotoxic Response to DNMT1-Targeted Therapy via Enhanced Viral Mimicry.

Polycomb repressive complex 2 (PRC2) has oncogenic and tumor-suppressive roles in cancer. There is clinical success of targeting this complex in PRC2-dependent cancers, but an unmet therapeutic need exists in PRC2-loss cancer. PRC2-inactivating mutations are a hallmark feature of high-grade malignant peripheral nerve sheath tumor (MPNST), an aggressive sarcoma with poor prognosis and no effective targeted therapy. Through RNAi screening in MPNST, we found that PRC2 inactivation increases sensitivity to genetic or small-molecule inhibition of DNA methyltransferase 1 (DNMT1), which results in enhanced cytotoxicity and antitumor response. Mechanistically, PRC2 inactivation amplifies DNMT inhibitor-mediated expression of retrotransposons, subsequent viral mimicry response, and robust cell death in part through a protein kinase R (PKR)-dependent double-stranded RNA sensor. Collectively, our observations posit DNA methylation as a safeguard against antitumorigenic cell-fate decisions in PRC2-loss cancer to promote cancer pathogenesis, which can be therapeutically exploited by DNMT1-targeted therapy. SIGNIFICANCE: PRC2 inactivation drives oncogenesis in various cancers, but therapeutically targeting PRC2 loss has remained challenging. Here we show that PRC2-inactivating mutations set up a tumor context-specific liability for therapeutic intervention via DNMT1 inhibitors, which leads to innate immune signaling mediated by sensing of derepressed retrotransposons and accompanied by enhanced cytotoxicity. See related commentary by Guil and Esteller, p. 2020. This article is highlighted in the In This Issue feature, p. 2007.