umors encompass complex cellular ecosystems of malignant and non-malignant cells, whose diversity and interactions affect cancer progression and drug response and resistance. Recent advances in single-cell genomics, especially single-cell RNA-Seq (scRNA-Seq), have transformed our ability to analyze tumors, revealing cell types, states, genetic diversity and interactions in the complex tumor ecosystem 1-6. Single-cell analysis of tumors is rapidly expanding, including the launch of a Human Tumor Atlas Network (HTAPP) as part of the Cancer Moonshot 7. Successful scRNA-Seq of clinical tumor specimens poses several challenges. First, it requires quick dissociation tailored to the tumor type, and involves enzymatic digestion, which can lead to loss of sensitive cells or changes in gene expression. Moreover, obtaining fresh tissue is time-sensitive and requires tight coordination

AbstractSingle cell genomics is essential to chart the complex tumor ecosystem. While single cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumor tissues, single nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each strategy requires modifications to fit the unique characteristics of different tissue and tumor types, posing a barrier to adoption. Here, we developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We tested eight tumor types of varying tissue and sample characteristics (resection, biopsy, ascites, and orthotopic patient-derived xenograft): lung cancer, metastatic breast cancer, ovarian cancer, melanoma, neuroblastoma, pediatric sarcoma, glioblastoma, pediatric high-grade glioma, and chronic lymphocytic leukemia. Analyzing 212,498 cells and nuclei from 39 clinical samples, we evaluated protocols by cell quality, recovery rate, and cellular composition. We optimized protocols for fresh tissue dissociation for different tumor types using a decision tree to account for the technical and biological variation between clinical samples. We established methods for nucleus isolation from OCT embedded and fresh-frozen tissues, with an optimization matrix varying mechanical force, buffer, and detergent. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types and intrinsic expression profiles, but at different proportions. Our work provides direct guidance across a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G/M pathway is the most effective approach in vivo for high-risk RMS.

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumorsuppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.

Cancer risk is highly variable in carriers of the common TP53-R337H founder allele, possibly due to the influence of modifier genes. Whole-genome sequencing identified a variant in the tumor suppressor XAF1 (E134*/Glu134Ter/rs146752602) in a subset of R337H carriers. Haplotype-defining variants were verified in 203 patients with cancer, 582 relatives, and 42,438 newborns. The compound mutant haplotype was enriched in patients with cancer, conferring risk for sarcoma (P = 0.003) and subsequent malignancies (P = 0.006). Functional analyses demonstrated that wild-type XAF1 enhances transactivation of wild-type and hypomorphic TP53 variants, whereas XAF1-E134* is markedly attenuated in this activity. We propose that cosegregation of XAF1-E134* and TP53-R337H mutations leads to a more aggressive cancer phenotype than TP53-R337H alone, with implications for genetic counseling and clinical management of hypomorphic TP53 mutant carriers.

SUMMARY

B7-H3 is actively being explored as an immunotherapy target for pediatric patients with solid tumors using monoclonal antibodies or T cells expressing chimeric antigen receptors (CARs). B7-H3-CARs containing a 41BB costimulatory domain are currently favored by several groups based on preclinical studies. In this study, we initially performed a detailed analysis of T cells expressing B7-H3-CARs with different hinge/transmembrane (CD8α versus CD28) and CD28 or 41BB costimulatory domains (CD8α/CD28, CD8α/41BB, CD28/CD28, CD28/41BB). Only subtle differences in effector function were observed between CAR T cell populations in vitro . However, CD8α/CD28-CAR T cells consistently outperformed other CAR T cell populations in three animal models, resulting in a significant survival advantage. We next explored whether adding 41BB signaling to CD8α/CD28-CAR T cells would further enhance effector function. Surprisingly, incorporating 41BB signaling into the CAR endodomain had detrimental effects, while expressing 41BBL on the surface of CD8α/CD28-CAR T cells enhanced their ability to kill tumor cells in repeat stimulation assays. Furthermore, 41BBL expression enhanced CD8α/CD28-CAR T cell expansion in vivo and improved antitumor activity in one of four evaluated models. Thus, our study highlights the intricate interplay between CAR hinge/transmembrane and costimulatory domains. Based on our study, we selected CD8α/CD28-CAR T cells expressing 41BBL for early phase clinical testing.

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