RESUMO
Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is frequently overexpressed in various carcinomas. We have developed chimeric antigen receptor (CAR) T cells specifically targeting EpCAM for the treatment of gastric cancer. This study sought to unravel the precise mechanisms by which tumors evade immune surveillance and develop resistance to CAR T cell therapy. Through a combination of whole-body CAR T cell imaging and single-cell multiomic analyses, we uncovered intricate interactions between tumors and tumor-infiltrating lymphocytes (TILs). In a gastric cancer model, tumor-infiltrating CD8 T cells exhibited both cytotoxic and exhausted phenotypes, while CD4 T cells were mainly regulatory T cells. A T cell receptor (TCR) clonal analysis provided evidence of CAR T cell proliferation and clonal expansion within resistant tumors, which was substantiated by whole-body CAR T cell imaging. Furthermore, single-cell transcriptomics showed that tumor cells in mice with refractory or relapsing outcomes were enriched for genes involved in major histocompatibility complex (MHC) and antigen presentation pathways, interferon-γ and interferon-α responses, mitochondrial activities, and a set of genes (e.g., CD74, IDO1, IFI27) linked to tumor progression and unfavorable disease prognoses. This research highlights an approach that combines imaging and multiomic methodologies to concurrently characterize the evolution of tumors and the differentiation of CAR T cells.
RESUMO
The limited number of targetable tumor-specific antigens and the immunosuppressive nature of the microenvironment within solid malignancies represent major barriers to the success of chimeric antigen receptor (CAR)-T cell therapies. Here, using epithelial cell adhesion molecule (EpCAM) as a model antigen, we used alanine scanning of the complementarity-determining region to fine-tune CAR affinity. This allowed us to identify CARs that could spare primary epithelial cells while still effectively targeting EpCAMhigh tumors. Although affinity-tuned CARs showed suboptimal antitumor activity in vivo, we found that inducible secretion of interleukin-12 (IL-12), under the control of the NFAT promoter, can restore CAR activity to levels close to that of the parental CAR. This strategy was further validated with another affinity-tuned CAR specific for intercellular adhesion molecule-1 (ICAM-1). Only in affinity-tuned CAR-T cells was NFAT activity stringently controlled and restricted to tumors expressing the antigen of interest at high levels. Our study demonstrates the feasibility of specifically gearing CAR-T cells towards recognition of solid tumors by combining inducible IL-12 expression and affinity-tuned CAR.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Animais , Camundongos , Interleucina-12/genética , Molécula de Adesão da Célula Epitelial , Imunoterapia Adotiva , Neoplasias/genética , Neoplasias/terapia , Antígenos de Neoplasias/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
Adoptive transfer of chimeric antigen receptor (CAR) T cells has demonstrated unparalleled responses in hematologic cancers, yet antigen escape and tumor relapse occur frequently. CAR T-cell therapy for patients with solid tumors faces even greater challenges due to the immunosuppressive tumor environment and antigen heterogeneity. Here, we developed a bispecific CAR to simultaneously target epithelial cell adhesion molecule (EpCAM) and intercellular adhesion molecule 1 (ICAM-1) to overcome antigen escape and to improve the durability of tumor responses. ICAM-1 is an adhesion molecule inducible by inflammatory cytokines and elevated in many types of tumors. Our study demonstrates superior efficacy of bispecific CAR T cells compared with CAR T cells targeting a single primary antigen. Bispecific CAR T achieved more durable antitumor responses in tumor models with either homogenous or heterogenous expression of EpCAM. We also showed that the activation of CAR T cells against EpCAM in tumors led to upregulation of ICAM-1, which rendered tumors more susceptible to ICAM-1 targeting by bispecific CAR T cells. Our strategy of additional targeting of ICAM-1 may have broad applications in augmenting the activity of CAR T cells against primary tumor antigens that are prone to antigen loss or downregulation.
Assuntos
Molécula de Adesão da Célula Epitelial/metabolismo , Imunoterapia Adotiva/métodos , Molécula 1 de Adesão Intercelular/metabolismo , Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Animais , Deriva e Deslocamento Antigênicos , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Molécula de Adesão da Célula Epitelial/genética , Humanos , Imunoterapia Adotiva/efeitos adversos , Molécula 1 de Adesão Intercelular/genética , Masculino , Camundongos , Neoplasias/imunologia , Neoplasias/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Small cell neuroendocrine cancers (SCNCs) are recalcitrant cancers arising from diverse primary sites that lack effective treatments. Using chemical genetic screens, we identified inhibition of ataxia telangiectasia and rad3 related (ATR), the primary activator of the replication stress response, and topoisomerase I (TOP1), nuclear enzyme that suppresses genomic instability, as synergistically cytotoxic in small cell lung cancer (SCLC). In a proof-of-concept study, we combined M6620 (berzosertib), first-in-class ATR inhibitor, and TOP1 inhibitor topotecan in patients with relapsed SCNCs. Objective response rate among patients with SCLC was 36% (9/25), achieving the primary efficacy endpoint. Durable tumor regressions were observed in patients with platinum-resistant SCNCs, typically fatal within weeks of recurrence. SCNCs with high neuroendocrine differentiation, characterized by enhanced replication stress, were more likely to respond. These findings highlight replication stress as a potentially transformative vulnerability of SCNCs, paving the way for rational patient selection in these cancers, now treated as a single disease.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Isoxazóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Pirazinas/farmacologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Idoso , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Replicação do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/genética , Instabilidade Genômica/genética , Humanos , Neoplasias Pulmonares/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Pequenas Células do Pulmão/metabolismoRESUMO
The discovery that enhancers are regulated transcription units, encoding eRNAs, has raised new questions about the mechanisms of their activation. Here, we report an unexpected molecular mechanism that underlies ligand-dependent enhancer activation, based on DNA nicking to relieve torsional stress from eRNA synthesis. Using dihydrotestosterone (DHT)-induced binding of androgen receptor (AR) to prostate cancer cell enhancers as a model, we show rapid recruitment, within minutes, of DNA topoisomerase I (TOP1) to a large cohort of AR-regulated enhancers. Furthermore, we show that the DNA nicking activity of TOP1 is a prerequisite for robust eRNA synthesis and enhancer activation and is kinetically accompanied by the recruitment of ATR and the MRN complex, followed by additional components of DNA damage repair machinery to the AR-regulated enhancers. Together, our studies reveal a linkage between eRNA synthesis and ligand-dependent TOP1-mediated nicking-a strategy exerting quantitative effects on eRNA expression in regulating AR-bound enhancer-dependent transcriptional programs.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Receptores Androgênicos/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Simples , Reparo do DNA , DNA Topoisomerases Tipo I/genética , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Humanos , Proteína Homóloga a MRE11 , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Chromosomes in PTEN deficient cells display both numerical as well as structural alterations including regional amplification. We found that PTEN deficient cells displayed a normal DNA damage response (DDR) as evidenced by the ionizing radiation (IR)-induced phosphorylation of Ataxia Telangiectasia Mutated (ATM) as well as its effectors. PTEN deficient cells also had no defect in Rad51 expression or DNA damage repair kinetics post irradiation. In contrast, caffeine treatment specifically increased IR-induced chromosome aberrations and mitotic index only in cells with PTEN, and not in cells deficient for PTEN, suggesting that their checkpoints were defective. Furthermore, PTEN-deficient cells were unable to maintain active spindle checkpoint after taxol treatment. Genomic instability in PTEN deficient cells could not be attributed to lack of PTEN at centromeres, since no interaction was detected between centromeric DNA and PTEN in wild type cells. These results indicate that PTEN deficiency alters multiple cell cycle checkpoints possibly leaving less time for DNA damage repair and/or chromosome segregation as evidenced by the increased structural as well as numerical alterations seen in PTEN deficient cells.
Assuntos
Ciclo Celular , Reparo do DNA , Instabilidade Genômica , PTEN Fosfo-Hidrolase/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Aberrações Cromossômicas , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Humanos , Raios Infravermelhos , Cariotipagem , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Paclitaxel/farmacologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Telômero/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Deciphering the epigenetic "code" remains a central issue in transcriptional regulation. Here, we report the identification of a JAMM/MPN(+) domain-containing histone H2A deubiquitinase (2A-DUB, or KIAA1915/MYSM1) specific for monoubiquitinated H2A (uH2A) that has permitted delineation of a strategy for specific regulatory pathways of gene activation. 2A-DUB regulates transcription by coordinating histone acetylation and deubiquitination, and destabilizing the association of linker histone H1 with nucleosomes. 2A-DUB interacts with p/CAF in a coregulatory protein complex, with its deubiquitinase activity modulated by the status of acetylation of nucleosomal histones. Consistent with this mechanistic role, 2A-DUB participates in transcriptional regulation events in androgen receptor-dependent gene activation, and the levels of uH2A are dramatically decreased in prostate tumors, serving as a cancer-related mark. We suggest that H2A ubiquitination represents a widely used mechanism for many regulatory transcriptional programs and predict that various H2A ubiquitin ligases/deubiquitinases will be identified for specific cohorts of regulated transcription units.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ubiquitinas/metabolismo , Acetilação/efeitos dos fármacos , Androgênios/farmacologia , Animais , Linhagem Celular , Cromatografia de Afinidade , Proteínas de Ligação a DNA/isolamento & purificação , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Humanos , Camundongos , Modelos Genéticos , Nucleossomos/efeitos dos fármacos , Nucleossomos/metabolismo , Fosforilação/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transativadores , Fatores de Transcrição/isolamento & purificação , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Proteases Específicas de UbiquitinaRESUMO
PTEN, a tumor suppressor gene, is frequently mutated in a variety of human tumors. In mice, monoallelic inactivation of this gene predisposes animals to neoplasia of multiple organs. Interestingly, Pten heterozygous mice develop bilateral hyperplasia of the adrenal medulla. In this report we demonstrate that these neoplasms are hormonally active pheochromocytomas that secrete increased amounts of bioactive catecholamines: norepinephrine and epinephrine. To test a possibility that PTEN might be one of the genes responsible for human sporadic pheochromocytoma, we performed mutation analysis of DNA obtained from tumors of 29 patients. However, direct sequencing of all nine exons of the PTEN gene, including the splice junctions, revealed no mutations. Examination of protein expression by immunohistochemistry using 8 normal adrenals and 11 sporadic pheochromocytomas showed no decrease in the PTEN protein expression in the tumor tissue, but upregulation of insulin-like growth factor II, a peptide implicated in growth of adrenal tissue, was observed in four cases (36%).
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Mutação , PTEN Fosfo-Hidrolase/genética , Feocromocitoma/genética , Animais , Sequência de Bases , Primers do DNA , Imuno-Histoquímica , CamundongosRESUMO
CHK1 is an essential kinase involved in the regulation of the cell cycle progression and preservation of genomic integrity. Inhibition of CHK1 leads to the accumulation of double-stranded DNA breaks. Loss of PTEN impairs CHK1-mediated checkpoint activation due to cytoplasmic sequestration of ubiquitinated CHK1. Here, we provide evidence that another consequence of reduced CHK1 function in PTEN deficient cells is the accumulation of double-stranded DNA breaks. Moreover, we show that the site of CHK1 ubiquitination (K274) is near the site of AKT phosphorylation (S280). Overall, these data demonstrate that lack of PTEN generates DNA damage due to inappropriate inactivation of CHK1.DNA damage due to the loss of PTEN is likely to stimulate tumor development.
Assuntos
Dano ao DNA/genética , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Quinase 1 do Ponto de Checagem , Regulação para Baixo , Camundongos , Dados de Sequência Molecular , Mutação/genética , Proteína Oncogênica v-akt/metabolismo , Ligação Proteica , Proteínas Quinases/química , Células-Tronco/efeitos da radiação , Ubiquitina/metabolismo , Raios UltravioletaRESUMO
Pten-/- cells display a partially defective checkpoint in response to ionizing radiation (IR). The checkpoint defect was traced to the ability of AKT to phosphorylate CHK1 at serine 280, since a nonphosphorylated mutant of CHK1 (S280A) complemented the checkpoint defect and restored CDC25A degradation. CHK1 phosphorylation at serine 280 led to covalent binding of 1 to 2 molecules of ubiquitin and cytoplasmic CHK1 localization. Primary breast carcinomas lacking PTEN expression and having elevated AKT phosphorylation had increased cytoplasmic CHK1 and displayed aneuploidy (p <0.005). We conclude that loss of PTEN and subsequent activation of AKT impair CHK1 through phosphorylation, ubiquitination, and reduced nuclear localization to promote genomic instability in tumor cells.