RESUMO
PURPOSE: Sarcoma encompasses a diverse group of cancers that are typically resistant to current therapies, including immune checkpoint blockade (ICB), and underlying mechanisms are poorly understood. The contexture of sarcomas limits generation of high-quality data using cutting-edge molecular profiling methods, such as single-cell RNA-sequencing, thus hampering progress in understanding these understudied cancers. EXPERIMENTAL DESIGN: Here, we demonstrate feasibility of producing multimodal single-cell genomics and whole-genome sequencing data from frozen tissues, profiling 75,716 cell transcriptomes of five undifferentiated pleomorphic sarcoma and three intimal sarcoma samples, including paired specimens from two patients treated with ICB. RESULTS: We find that genomic diversity decreases in patients with response to ICB, and, in unbiased analyses, identify cancer cell programs associated with therapy resistance. Although interactions of tumor-infiltrating T lymphocytes within the tumor ecosystem increase in ICB responders, clonal expansion of CD8+ T cells alone was insufficient to predict drug responses. CONCLUSIONS: This study provides a framework for studying rare tumors and identifies salient and treatment-associated cancer cell intrinsic and tumor microenvironmental features in sarcomas.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico , Sarcoma , Análise de Célula Única , Humanos , Sarcoma/genética , Sarcoma/tratamento farmacológico , Sarcoma/patologia , Sarcoma/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Análise de Célula Única/métodos , Resistencia a Medicamentos Antineoplásicos/genética , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Microambiente Tumoral/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Feminino , Perfilação da Expressão Gênica , Masculino , Transcriptoma , Biomarcadores Tumorais/genética , Pessoa de Meia-Idade , Sequenciamento Completo do GenomaRESUMO
Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Here, we perform massively parallel base-editing screens to generate thousands of variants at gene loci annotated with known or potential clinical relevance. We discover a broad landscape of putative gain-of-function (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and the gene encoding its regulatory subunit, PIK3R1, LCK, SOS1, AKT1 and RHOA. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific to a melanoma epitope or in different generations of CD19 chimeric antigen receptor (CAR) T cells demonstrates that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, we show that generations of CD19 CAR T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production and leukemia cell killing, including when benchmarked against other recent strategies.
RESUMO
Chromosome-containing micronuclei are a hallmark of aggressive cancers. Micronuclei frequently undergo irreversible collapse, exposing their enclosed chromatin to the cytosol. Micronuclear rupture catalyzes chromosomal rearrangements, epigenetic abnormalities, and inflammation, yet mechanisms safeguarding micronuclear integrity are poorly understood. In this study, we found that mitochondria-derived reactive oxygen species (ROS) disrupt micronuclei by promoting a noncanonical function of charged multivesicular body protein 7 (CHMP7), a scaffolding protein for the membrane repair complex known as endosomal sorting complex required for transport III (ESCRT-III). ROS retained CHMP7 in micronuclei while disrupting its interaction with other ESCRT-III components. ROS-induced cysteine oxidation stimulated CHMP7 oligomerization and binding to the nuclear membrane protein LEMD2, disrupting micronuclear envelopes. Furthermore, this ROS-CHMP7 pathological axis engendered chromosome shattering known to result from micronuclear rupture. It also mediated micronuclear disintegrity under hypoxic conditions, linking tumor hypoxia with downstream processes driving cancer progression.