Genetic Screen in a Pre-Clinical Model of Sarcoma Development Defines Drivers and Therapeutic Vulnerabilities.

PURPOSE: High-grade complex karyotype sarcomas are a heterogeneous group of tumors with a uniformly poor prognosis. Within complex karyotype sarcomas, there are innumerable genetic changes but identifying those that are clinically relevant has been challenging. EXPERIMENTAL DESIGN: To address this, we utilized a pooled genetic screening approach, informed by TCGA data, to identify key drivers and modifiers of sarcoma development that were validated in vivo. RESULTS: YAP1 and wildtype KRAS were validated as drivers and transformed human mesenchymal stem cells into two distinct sarcoma subtypes, undifferentiated pleomorphic sarcoma (UPS) and myxofibrosarcoma (MFS), respectively. A subset of tumors driven by CDK4 and PIK3CA reflected leiomyosarcoma (LMS) and osteosarcoma (OS) demonstrating the plasticity of this approach and the potential to investigate sarcoma subtype heterogeneity. All generated tumors histologically reflected human sarcomas and had increased aneuploidy as compared to simple karyotype sarcomas. Comparing differential gene expression of TCGA samples to model data identified increased oxidative phosphorylation signaling in YAP1 tumors. Treatment of a panel of soft tissue sarcomas with a combination of YAP1 and oxidative phosphorylation inhibitors led to significantly decreased viability. CONCLUSIONS: Transcriptional co-analysis of TCGA patient samples to YAP1 and KRAS model tumors support that these sarcoma subtypes lie along a spectrum of disease and adds guidance for further transcriptome-based refinement of sarcoma subtyping. This approach can be used to begin to understand pathways and mechanisms driving human sarcoma development, the relationship between sarcoma subtypes and to identify and validate new therapeutic vulnerabilities for this aggressive and heterogeneous disease.

PGC-1α drives small cell neuroendocrine cancer progression towards an ASCL1-expressing subtype with increased mitochondrial capacity.

Adenocarcinomas from multiple tissues can converge to treatment-resistant small cell neuroendocrine (SCN) cancers comprised of ASCL1, POU2F3, NEUROD1, and YAP1 subtypes. We investigated how mitochondrial metabolism influences SCN cancer (SCNC) progression. Extensive bioinformatics analyses encompassing thousands of patient tumors and human cancer cell lines uncovered enhanced expression of PGC-1α, a potent regulator of mitochondrial oxidative phosphorylation (OXPHOS), across several SCNC types. PGC-1α correlated tightly with increased expression of the lineage marker ASCL1 through a positive feedback mechanism. Analyses using a human prostate tissue-based SCN transformation system showed that the ASCL1 subtype has heightened PGC-1α expression and OXPHOS activity. PGC-1α inhibition diminished OXPHOS, reduced SCNC cell proliferation, and blocked SCN prostate tumor formation. PGC-1α overexpression enhanced OXPHOS, tripled the SCN prostate tumor formation rate, and promoted commitment to the ASCL1 lineage. These findings reveal the metabolic heterogeneity among SCNC subtypes and identify PGC-1α-induced OXPHOS as a regulator of SCNC lineage plasticity.

Defining T cell receptor repertoires using nanovial-based binding and functional screening.

The ability to selectively bind to antigenic peptides and secrete effector molecules can define rare and low-affinity populations of cells with therapeutic potential in emerging T cell receptor (TCR) immunotherapies. We leverage cavity-containing hydrogel microparticles, called nanovials, each coated with peptide-major histocompatibility complex (pMHC) monomers to isolate antigen-reactive T cells. T cells are captured and activated by pMHCs inducing the secretion of effector molecules including IFN-γ and granzyme B that are accumulated on nanovials, allowing sorting based on both binding and function. The TCRs of sorted cells on nanovials are sequenced, recovering paired αß-chains using microfluidic emulsion-based single-cell sequencing. By labeling nanovials having different pMHCs with unique oligonucleotide-barcodes and secretions with oligo-barcoded detection antibodies, we could accurately link TCR sequences to specific targets and rank each TCR based on the corresponding cell's secretion level. Using the technique, we identified an expanded repertoire of functional TCRs targeting viral antigens with high specificity and found rare TCRs with activity against cancer-specific splicing-enhanced epitopes.