Anti-TIGIT antibody improves PD-L1 blockade through myeloid and Treg cells.

Tiragolumab, an anti-TIGIT antibody with an active IgG1κ Fc, demonstrated improved outcomes in the phase 2 CITYSCAPE trial (ClinicalTrials.gov: NCT03563716 ) when combined with atezolizumab (anti-PD-L1) versus atezolizumab alone1. However, there remains little consensus on the mechanism(s) of response with this combination2. Here we find that a high baseline of intratumoural macrophages and regulatory T cells is associated with better outcomes in patients treated with atezolizumab plus tiragolumab but not with atezolizumab alone. Serum sample analysis revealed that macrophage activation is associated with a clinical benefit in patients who received the combination treatment. In mouse tumour models, tiragolumab surrogate antibodies inflamed tumour-associated macrophages, monocytes and dendritic cells through Fcγ receptors (FcγR), in turn driving anti-tumour CD8+ T cells from an exhausted effector-like state to a more memory-like state. These results reveal a mechanism of action through which TIGIT checkpoint inhibitors can remodel immunosuppressive tumour microenvironments, and suggest that FcγR engagement is an important consideration in anti-TIGIT antibody development.

Immunomodulatory effects and improved outcomes with cisplatin- versus carboplatin-based chemotherapy plus atezolizumab in urothelial cancer.

In metastatic urothelial cancer (mUC), cisplatin versus carboplatin leads to durable disease control in a subset of patients. The IMvigor130 trial reveals more favorable effects with atezolizumab combined with gemcitabine and cisplatin (GemCis) versus gemcitabine and carboplatin (GemCarbo). This study investigates the immunomodulatory effects of cisplatin as a potential explanation for these observations. Our findings indicate that improved outcomes with GemCis versus GemCarbo are primarily observed in patients with pretreatment tumors exhibiting features of restrained adaptive immunity. In addition, GemCis versus GemCarbo ± atezolizumab induces transcriptional changes in circulating immune cells, including upregulation of antigen presentation and T cell activation programs. In vitro experiments demonstrate that cisplatin, compared with carboplatin, exerts direct immunomodulatory effects on cancer cells, promoting dendritic cell activation and antigen-specific T cell killing. These results underscore the key role of immune modulation in cisplatin's efficacy in mUC and highlight the importance of specific chemotherapy backbones in immunotherapy combination regimens.

Anti-inflammatory microRNA-146a protects mice from diet-induced metabolic disease.

Identifying regulatory mechanisms that influence inflammation in metabolic tissues is critical for developing novel metabolic disease treatments. Here, we investigated the role of microRNA-146a (miR-146a) during diet-induced obesity in mice. miR-146a is reduced in obese and type 2 diabetic patients and our results reveal that miR-146a-/- mice fed a high-fat diet (HFD) have exaggerated weight gain, increased adiposity, hepatosteatosis, and dysregulated blood glucose levels compared to wild-type controls. Pro-inflammatory genes and NF-κB activation increase in miR-146a-/- mice, indicating a role for this miRNA in regulating inflammatory pathways. RNA-sequencing of adipose tissue macrophages demonstrated a role for miR-146a in regulating both inflammation and cellular metabolism, including the mTOR pathway, during obesity. Further, we demonstrate that miR-146a regulates inflammation, cellular respiration and glycolysis in macrophages through a mechanism involving its direct target Traf6. Finally, we found that administration of rapamycin, an inhibitor of mTOR, was able to rescue the obesity phenotype in miR-146a-/- mice. Altogether, our study provides evidence that miR-146a represses inflammation and diet-induced obesity and regulates metabolic processes at the cellular and organismal levels, demonstrating how the combination of diet and miRNA genetics influences obesity and diabetic phenotypes.

Antitumor immunity is defective in T cell-specific microRNA-155-deficient mice and is rescued by immune checkpoint blockade.

MicroRNA-155 (miR-155) regulates antitumor immune responses. However, its specific functions within distinct immune cell types have not been delineated in conditional KO mouse models. In this study, we investigated the role of miR-155 specifically within T cells during the immune response to syngeneic tumors. We found that miR-155 expression within T cells is required to limit syngeneic tumor growth and promote IFNγ production by T cells within the tumor microenvironment. Consequently, we found that miR-155 expression by T cells is necessary for proper tumor-associated macrophage expression of IFNγ-inducible genes. We also found that immune checkpoint-blocking (ICB) antibodies against programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) restored antitumor immunity in miR-155 T cell-conditional KO mice. We noted that these ICB antibodies rescued the levels of IFNγ-expressing T cells, expression of multiple activation and effector genes expressed by tumor-infiltrating CD8+ and CD4+ T cells, and tumor-associated macrophage activation. Moreover, the ICB approach partially restored expression of several derepressed miR-155 targets in tumor-infiltrating, miR-155-deficient CD8+ T cells, suggesting that miR-155 and ICB regulate overlapping pathways to promote antitumor immunity. Taken together, our findings highlight the multifaceted role of miR-155 in T cells, in which it promotes antitumor immunity. These results suggest that the augmentation of miR-155 expression could be used to improve anticancer immunotherapies.

Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 single T cells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8+ T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8+ T cells and Tregs and represses the CD8+ T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers.

miR-155 promotes FLT3-ITD-induced myeloproliferative disease through inhibition of the interferon response.

FLT3-ITD+ acute myeloid leukemia (AML) accounts for ∼25% of all AML cases and is a subtype that carries a poor prognosis. microRNA-155 (miR-155) is specifically overexpressed in FLT3-ITD+ AML compared with FLT3 wild-type (FLT3-WT) AML and is critical for the growth of FLT3-ITD+ AML cells in vitro. However, miR-155's role in regulating FLT3-ITD-mediated disease in vivo remains unclear. In this study, we used a genetic mouse model to determine whether miR-155 influences the development of FLT3-ITD-induced myeloproliferative disease. Results indicate that miR-155 promotes FLT3-ITD-induced myeloid expansion in the bone marrow, spleen, and peripheral blood. Mechanistically, miR-155 increases proliferation of the hematopoietic stem and progenitor cell compartments by reducing the growth-inhibitory effects of the interferon (IFN) response, and this involves targeting of Cebpb. Consistent with our observations in mice, primary FLT3-ITD+ AML clinical samples have significantly higher miR-155 levels and a lower IFN response compared with FLT3-WT AML samples. Further, inhibition of miR-155 in FLT3-ITD+ AML cell lines using CRISPR/Cas9, or primary FLT3-ITD+ AML samples using locked nucleic acid antisense inhibitors, results in an elevated IFN response and reduces colony formation. Altogether, our data reveal that miR-155 collaborates with FLT3-ITD to promote myeloid cell expansion in vivo and that this involves a multitarget mechanism that includes repression of IFN signaling.

Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth.

Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.

Exosome-delivered microRNAs modulate the inflammatory response to endotoxin.

MicroRNAs regulate gene expression posttranscriptionally and function within the cells in which they are transcribed. However, recent evidence suggests that microRNAs can be transferred between cells and mediate target gene repression. We find that endogenous miR-155 and miR-146a, two critical microRNAs that regulate inflammation, are released from dendritic cells within exosomes and are subsequently taken up by recipient dendritic cells. Following uptake, exogenous microRNAs mediate target gene repression and can reprogramme the cellular response to endotoxin, where exosome-delivered miR-155 enhances while miR-146a reduces inflammatory gene expression. We also find that miR-155 and miR-146a are present in exosomes and pass between immune cells in vivo, as well as demonstrate that exosomal miR-146a inhibits while miR-155 promotes endotoxin-induced inflammation in mice. Together, our findings provide strong evidence that endogenous microRNAs undergo a functional transfer between immune cells and constitute a mechanism of regulating the inflammatory response.

Genome-wide transcriptome profiling of homologous recombination DNA repair.

Homologous recombination (HR) repair deficiency predisposes to cancer development, but also sensitizes cancer cells to DNA damage-inducing therapeutics. Here we identify an HR defect (HRD) gene signature that can be used to functionally assess HR repair status without interrogating individual genetic alterations in cells. By using this HRD gene signature as a functional network analysis tool, we discover that simultaneous loss of two major tumour suppressors BRCA1 and PTEN extensively rewire the HR repair-deficient phenotype, which is found in cells with defects in either BRCA1 or PTEN alone. Moreover, the HRD gene signature serves as an effective drug discovery platform to identify agents targeting HR repair as potential chemo/radio sensitizers. More importantly, this HRD gene signature is able to predict clinical outcomes across multiple cancer lineages. Our findings, therefore, provide a molecular profile of HR repair to assess its status at a functional network level, which can provide both biological insights and have clinical implications in cancer.

Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage.

Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.

BRIT1 regulates p53 stability and functions as a tumor suppressor in breast cancer.

In humans, the gene encoding the BRCA1 C terminus-repeat inhibitor of human telomerase expression 1 (BRIT1) protein is located on chromosome 8p23.1, a region implicated in the development of several malignancies, including breast cancer. Previous studies by our group and others suggested that BRIT1 might function as a novel tumor suppressor. Thus, identifying the molecular mechanisms that underlie BRIT1's tumor suppressive function is important to understand cancer etiology and to identify effective therapeutic strategies for BRIT1-deficient tumors. We thus investigated the role of BRIT1 as a tumor suppressor in breast cancer by using genetic approaches. We discovered that BRIT1 functions as a post-transcriptional regulator of p53 expression. BRIT1 regulates p53 protein stability through blocking murine double minute 2-mediated p53 ubiquitination. To fully demonstrate the role of BRIT1 as a tumor suppressor, we depleted BRIT1 in normal breast epithelial cells. We found that knockdown of BRIT1 caused the oncogenic transformation of normal mammary epithelial cells. Furthermore, ectopic expression of BRIT1 effectively suppressed breast cancer cell proliferation and colony formation in vitro and tumor growth in vivo. Taken together, our study provides new insights into the biological functions of BRIT1 as a tumor suppressor in human breast cancer.

Epistasis between microRNAs 155 and 146a during T cell-mediated antitumor immunity.

An increased understanding of antitumor immunity is necessary for improving cell-based immunotherapies against human cancers. Here, we investigated the roles of two immune system-expressed microRNAs (miRNAs), miR-155 and miR-146a, in the regulation of antitumor immune responses. Our results indicate that miR-155 promotes and miR-146a inhibits interferon γ (IFNγ) responses by T cells and reduces solid tumor growth in vivo. Using a double-knockout (DKO) mouse strain deficient in both miR-155 and miR-146a, we have also identified an epistatic relationship between these two miRNAs. DKO mice had defective T cell responses and tumor growth phenotypes similar to miR-155(-/-) mice. Further analysis of the T cell compartment revealed that miR-155 modulates IFNγ expression through a mechanism involving repression of Ship1. Our work reveals critical roles for miRNAs in the reciprocal regulation of CD4(+) and CD8(+) T cell-mediated antitumor immunity and demonstrates the dominant nature of miR-155 during its promotion of immune responses.

ZNF668 functions as a tumor suppressor by regulating p53 stability and function in breast cancer.

Genome-wide sequencing studies in breast cancer have recently identified frequent mutations in the zinc finger protein 668 (ZNF668), the function of which is undefined. Here, we report that ZNF668 is a nucleolar protein that physically interacts with and regulates p53 and its negative regulator MDM2. Through MDM2 binding, ZNF668 regulated autoubiquitination of MDM2 and its ability to mediate p53 ubiquitination and degradation. ZNF668 deficiency also impaired DNA damage-induced stabilization of p53. RNA interference-mediated knockdown of ZNF668 was sufficient to transform normal mammary epithelial cells. ZNF668 effectively suppressed breast cancer cell proliferation in vitro and tumorigenicity in vivo. Taken together, our studies identify ZNF668 as a novel breast tumor suppressor gene that functions in regulating p53 stability.

BRIT1/MCPH1 links chromatin remodelling to DNA damage response.

To detect and repair damaged DNA, DNA-damage-response proteins need to overcome the barrier of condensed chromatin to gain access to DNA lesions. ATP-dependent chromatin remodelling is one of the fundamental mechanisms used by cells to relax chromatin in DNA repair. However, the mechanism mediating their recruitment to DNA lesions remains largely unknown. BRIT1 (also known as MCPH1) is an early DNA-damage-response protein that is mutated in human primary microcephaly. Here we report a previously unknown function of BRIT1 as a regulator of the ATP-dependent chromatin remodelling complex SWI-SNF in DNA repair. After damage to DNA, BRIT1 increases its interaction with SWI-SNF through ATM/ATR-dependent phosphorylation on the BAF170 subunit. This increase in binding affinity provides a means by which SWI-SNF can be specifically recruited to and maintained at DNA lesions. Loss of BRIT1 causes impaired chromatin relaxation as a result of decreased association of SWI-SNF with chromatin. This explains the decreased recruitment of repair proteins to DNA lesions and the reduced efficiency of repair in BRIT1-deficient cells, resulting in impaired cell survival after DNA damage. Our findings therefore identify BRIT1 as a key molecule that links chromatin remodelling with response to DNA damage in the control of DNA repair, and its dysfunction contributes to human disease.

Rak functions as a tumor suppressor by regulating PTEN protein stability and function.

Expression of the PTEN tumor suppressor is frequently lost in breast cancer in the absence of mutation or promoter methylation through as yet undetermined mechanisms. In this study, we demonstrate that the Rak tyrosine kinase physically interacts with PTEN and phosphorylates PTEN on Tyr336. Knockdown of Rak enhanced the binding of PTEN to its E3 ligase NEDD4-1 and promoted PTEN polyubiquitination, leading to PTEN protein degradation. Notably, ectopic expression of Rak effectively suppressed breast cancer cell proliferation, invasion, and colony formation in vitro and tumor growth in vivo. Furthermore, Rak knockdown was sufficient to transform normal mammary epithelial cells. Therefore, Rak acts as a bona fide tumor suppressor gene through the mechanism of regulating PTEN protein stability and function.

In vitro differentiation of hepatic progenitor cells from mouse embryonic stem cells induced by sodium butyrate.

Recently it was shown that embryonic stem (ES) cells could differentiate into hepatocytes both in vitro and in vivo, however, prospective hepatic progenitor cells have not yet been isolated and characterized from ES cells. Here we presented a novel 4-step procedure for the differentiation of mouse ES cells into hepatic progenitor cells and then hepatocytes. The differentiated hepatocytes were identified by morphological, biochemical, and functional analyses. The hepatic progenitor cells were isolated from the cultures after the withdrawal of sodium butyrate, which was characterized by scant cytoplasm, ovoid nuclei, the ability of rapid proliferation, expression of a series of hepatic progenitor cell markers, and the potential of differentiation into hepatocytes and bile duct-like cells under the proper conditions that favor hepatocyte and bile epithelial differentiation. The differentiation of hepatocytes from hepatic progenitor cells was characterized by a number of hepatic cell markers including albumin secretion, upregulated transcription of glucose-6-phosphatase and tyrosine aminotransferase, and functional phenotypes such as glycogen storage. The results from our experiments demonstrated that ES cells could differentiate into a novel bipotential hepatic progenitor cell and mature into hepatocytes with typical morphological, phenotypic and functional characteristics, which provides an useful model for the studies of key events during early liver development and a potential source of transplantable cells for cell-replacement therapies.

Generation of embryoid bodies from mouse embryonic stem cells cultured on STO feeder cells.

Embryoid bodies, which are similar to post-implantation egg-cylinder stage embryos, provide a model for the study of embryo development and stem cell differentiation. We describe here a novel method for generating embryoid bodies from murine embryonic stem (ES) cells cultured on the STO feeder layer. The ES cells grew into compact aggregates in the first 3 days of coculture, then became simple embryoid bodies (EBs) possessing primitive endoderm on the outer layer. They finally turned into cystic embryoid bodies after being transferred to Petri dishes for 1-3 days. Evaluation of the EBs in terms of morphology and differentiating potential indicates that they were typical in structure and could generate cells derived from the three germ layers. The results show that embryoid bodies can form not only in suspension culture but also directly from ES cells cultured on the STO feeder layer.