Journal of Clinical Investigation最新文献

Despite the revolutionary achievements of chimeric antigen receptor (CAR) T cell therapy in treating cancers, especially leukemia, several key challenges still limit its therapeutic efficacy. Of particular relevance is the relapse of cancer in large part, as a result of exhaustion and short persistence of CAR-T cells in vivo. IL-2-inducible T cell kinase (ITK) is a critical modulator of the strength of T-cell receptor (TCR) signaling, while its role in CAR signaling is unknown. By electroporation of clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) ribonucleoprotein (RNP) complex into CAR-T cells, we successfully deleted ITK in CD19-CAR-T cells with high efficiency. Bulk and single-cell RNA sequencing (scRNA-seq) analyses revealed down-regulation of exhaustion and up-regulation of memory gene signatures in ITK-deficient CD19-CAR-T cells. Our results further demonstrated a significant reduction of T cell exhaustion and enhancement of T cell memory, with significant improvement of CAR-T cell expansion and persistence both in vitro and in vivo. Moreover, ITK-deficient CD19-CAR-T cells showed better control of tumor relapse. Our work provides a promising strategy of targeting ITK to develop sustainable CAR-T products for clinical use.

Cardiac endothelial cells are essential for heart development, and disruption of this process can lead to congenital heart disease (CHD). However, how microRNAs influence cardiac endothelial cells in CHD remains unclear. This study identified elevated microRNA-187 (miR-187) expression in embryonic heart endothelial cells from CHD fetuses. Using a conditional knockin model, we showed that increased miR-187 levels in embryonic endothelial cells induce CHD in homozygous fetal mice, closely mirroring human CHD. Mechanistically, miR-187 targets NIPBL, which is responsible for recruiting the cohesin complex and facilitating chromatin accessibility. Consequently, the endothelial cell-specific upregulation of miR-187 inhibited NIPBL, leading to reduced chromatin accessibility and impaired gene expression, which hindered endothelial cell development and ultimately caused heart septal defects and reduced heart size both in vitro and in vivo. Importantly, exogenous miR-187 expression in human cardiac organoids mimicked developmental defects in the cardiac endothelial cells, and this was reversible by NIPBL replenishment. Our findings establish the miR-187/NIPBL axis as a potent regulator that inhibits cardiac endothelial cell development by attenuating the transcription of numerous endothelial genes, with our mouse and human cardiac organoid models effectively replicating severe defects from minor perturbations. This discovery suggests that targeting the miR-187/NIPBL pathway could offer a promising therapeutic approach for CHD.

The Hippo signaling pathway plays a key role in tumorigenesis in different cancer types. We investigated the role of the Hippo "effector" YAP1 on the tumor immune microenvironment (TIME) of urothelial carcinoma of bladder (UCB) and evaluated the efficacy of immunotherapy in the context of YAP1 signaling. We performed numerous in vitro and in vivo experiments to determine the role of YAP1 using genetic and pharmacological attenuation of YAP1 activity. Briefly, RNA sequencing was carried out with mice and human cell lines to identify novel YAP1-regulated downstream targets unbiasedly. We then experimentally confirmed that YAP1 regulates the TIME through the IL-6/STAT3 signaling pathway and varied C-X-C motif chemokine regulation. We analyzed several human sample sets to explore the TIME status in the context of YAP1 expression. Our data indicate that YAP1 attenuation decreases M2 macrophages and MDSCs in the TIME compared to YAP1 expressing cells. In summary, this study provides insights on YAP1 signaling as a driver for cancer stemness and an inducer of immunosuppressive TIME. Moreover, the therapeutic efficacy of YAP1 attenuation indicates that combined blockade of YAP1 and immune checkpoints may yield clinical value for treating UCB patients.

Dysregulations of epithelial-immune interactions frequently culminate in chronic inflammatory diseases of the skin, lungs, kidneys, and gastrointestinal tract. Yet, the intraepithelial processes which initiate and perpetuate inflammation in these organs are poorly understood. Here, by utilizing redox lipidomics we identified ferroptosis-associated peroxidation of polyunsaturated phosphatidylethanolamines in the epithelia of patients with asthma, cystic fibrosis, psoriasis and renal failure. Focusing on psoriasis as a disease model, we used high-resolution mass spectrometry imaging and identified keratin 14 (K14)-expressing keratinocytes executing a ferroptotic death program in human psoriatic skin. Psoriatic phenotype with characteristic Th1/Th17 skin and extracutaneous immune responses was initiated and maintained in a murine model designed to actuate ferroptosis in a fraction of K14+ glutathione peroxidase 4 (Gpx4)-deficient epidermal keratinocytes. Importantly, an anti-ferroptotic agent, Liproxstatin-1, was as effective as clinically relevant biologic IL-12/IL-23/TNFα-targeting therapies or the depletion of T cells in completely abrogating molecular, biochemical and morphologic features of psoriasis. As ferroptosis in select epidermal keratinocytes triggers and sustains a pathologic psoriatic multi-organ inflammatory circuit, we suggest that strategies targeting ferroptosis, or its causes, may be effective in preventing or ameliorating a variety of chronic inflammatory diseases.

Activating transcription factor 6 (Atf6) is a key regulator of the unfolded protein response (UPR) and is important for endoplasmic reticulum (ER) function and protein homeostasis in metazoan cells. Patients carrying loss-of-function ATF6 disease alleles develop the cone dysfunction disorder, achromatopsia. The impact of loss of ATF6 function on other cell types, organs, and diseases in people remains unclear. Here, we reported that progressive sensorineural hearing loss was a notable complaint in some patients carrying ATF6 disease alleles and that Atf6-/- mice also showed progressive auditory deficits affecting both genders. In mice with hearing deficits, we found disorganized stereocilia on hair cells and focal loss of outer hair cells. Transcriptomic analysis of Atf6-/- cochleae revealed marked induction of UPR, especially through the PERK arm. These findings identify ATF6 as an essential regulator of cochlear health and function. Furthermore, they supported that ATF6 inactivation in people causes progressive sensorineural hearing loss as part of a blindness-deafness genetic syndrome targeting hair cells and cone photoreceptors. Lastly, our genetic findings support ER stress as an important pathomechanism underlying cochlear damage and hearing loss with clinical implications for patient lifestyle modifications that minimize environmental/physiologic sources of ER stress to the ear.

Recent progress in cancer cell-based therapies has led to effective targeting and robust immune responses against cancer. However, the inherent safety risks of using live cancer cells necessitate the creation of an optimized safety switch without hindering the efficacy of immunotherapy. The existing safety switches typically induce tolerogenic cell death, potentially leading to an immunosuppressive tumor immune microenvironment (TIME), which is counterproductive to the goals of immunotherapy. Here, we developed and characterized an inducible RIPK3-driven necroptotic system that serves as a dual function of safety switch as well as inducing immunogenic cell death which in turn stimulates antitumor immune responses. We showed that activating RIPK3 safety switch triggered immunogenic responses marked by an increased release of adenosine triphosphate (ATP) and damage-associated molecular patterns (DAMPs). Compared to other existing safety switches, incorporating RIPK3 system inhibited tumor growth, improved survival outcomes in tumor-bearing mice, and fostered long-term antitumor immunity. Moreover, RIPK3 system reinvigorated the TIME by promoting dendritic cell (DC) maturation, polarizing the macrophages towards the M1 phenotype, and reducing the exhaustion of CD4+ and CD8+ T lymphocytes. Our study highlights the dual role of RIPK3-driven necroptotic system in improving the safety and efficacy of cancer cell-based therapy, with broader implications for cellular therapies.

Phenotypic plasticity is a hallmark of cancer and increasingly realized as a mechanism of resistance to androgen receptor (AR)-targeted therapy. Now that many prostate cancer (PCa) patients are treated upfront with AR-targeted agents, it's critical to identify actionable mechanisms that drive phenotypic plasticity, to prevent the emergence of resistance. We showed that loss of tristetraprolin (TTP, gene ZFP36) increased NF-κB activation, and was associated with higher rates of aggressive disease and early recurrence in primary PCa. We also examined the clinical and biological impact of ZFP36 loss with co-loss of PTEN, a known driver of PCa. Analysis of multiple independent primary PCa cohorts demonstrated that PTEN and ZFP36 co-loss was associated with increased recurrence risk. Engineering prostate-specific Zfp36 deletion in vivo, induced prostatic intraepithelial neoplasia, and, with Pten co-deletion, resulted in rapid progression to castration-resistant adenocarcinoma. Zfp36 loss altered the cell state driven by Pten loss, demonstrated by enrichment of EMT, inflammation, TNFα/NF-κB, IL6-JAK/STAT3 gene sets. Additionally, our work revealed that ZFP36 loss also induced enrichment of multiple gene sets involved in mononuclear cell migration, chemotaxis, and proliferation. Use of the NF-κB inhibitor, dimethylaminoparthenolide (DMAPT) induced marked therapeutic responses in tumors with PTEN and ZFP36 co-loss and reversed castration resistance.

The glioblastoma (GBM) microenvironment is enriched in immunosuppressive factors that potently interfere with the function of cytotoxic T lymphocytes. Cancer cells can directly impact the immune system, but the mechanisms driving these interactions are not completely clear. Here we demonstrate that the polyamine metabolite spermidine (SPD) is elevated in the GBM tumor microenvironment. Exogenous administration of SPD drives tumor aggressiveness in an immune-dependent manner in pre-clinical mouse models via reduction of CD8+ T cell frequency and reduced cytotoxic function. Knockdown of ornithine decarboxylase, the rate-limiting enzyme in spermidine synthesis, did not impact cancer cell growth in vitro but did result in extended survival. Furthermore, glioblastoma patients with a more favorable outcome had a significant reduction in spermidine compared to patients with a poor prognosis. Our results demonstrate that spermidine functions as a cancer cell-derived metabolite that drives tumor progression by reducing CD8+ T cell number and function.

Severe congenital neutropenia (SCN) is frequently associated with dominant point mutations in ELANE, the gene encoding neutrophil elastase (NE). Chronic administration of granulocyte colony-stimulating factor (G-CSF) is a first-line treatment of ELANE-mutant (ELANEmut) SCN. However, some ELANEmut patients including patients with ELANE start codon mutations do not respond to G-CSF. Here, through directed granulopoiesis of gene-edited isogenic normal and patient-derived iPSCs, we demonstrate that ELANE start codon mutations suffice to induce G-CSF resistant granulocytic precursor cell death and refractory SCN. ELANE start codon mutated neutrophil precursors express predominantly nuclear N-terminal truncated alternate NE. Unlike G-CSF sensitive ELANE mutations that induce endoplasmic reticulum and unfolded protein response stress, we found that the mutation of the ELANE translation initiation codon resulted in NE aggregates and activated pro-apoptotic aggrephagy as determined by downregulated BAG1 expression, decreased BAG1/BAG3 ratio, NE co-localization with BAG3, and localized expression of autophagic LC3B. We found that SERF1, an RNA-chaperone protein, known to localize in misfolded protein aggregates in neurodegenerative diseases, was highly upregulated and interacted with cytoplasmic NE of mutant neutrophil precursors. Silencing of SERF1 enhanced survival and differentiation of iPSC-derived neutrophil precursors, restoring their responsiveness to G-CSF. These observations provide a mechanistic insight of G-CSF-resistant ELANEmut SCN, revealing targets for therapeutic intervention.

Glioblastoma (GBM), an aggressive brain malignancy with a cellular hierarchy dominated by GBM stem cells (GSCs), evades antitumor immunity through mechanisms that remain incompletely understood. Like most cancers, GBMs undergo metabolic reprogramming toward glycolysis to generate lactate. Here, we show that lactate production by patient-derived GSCs and microglia/macrophages induces tumor cell epigenetic reprogramming through histone lactylation, an activating modification that leads to immunosuppressive transcriptional programs and suppression of phagocytosis via transcriptional upregulation of CD47, a "don't eat me" signal, in GBM cells. Leveraging these findings, pharmacologic targeting of lactate production augments efficacy of anti-CD47 therapy. Mechanistically, lactylated histone interacts with the heterochromatin component chromobox protein homolog 3 (CBX3). Although CBX3 does not possess direct lactyltransferase activity, CBX3 binds histone acetyltransferase (HAT) EP300 to induce increased EP300 substrate specificity toward lactyl-CoA and a transcriptional shift toward an immunosuppressive cytokine profile. Targeting CBX3 inhibits tumor growth by both tumor cell-intrinsic mechanisms and increased tumor cell phagocytosis. Collectively, these results suggest that lactate mediates metabolism-induced epigenetic reprogramming in GBM that contributes to CD47-dependent immune evasion, which can be leveraged to augment efficacy of immuno-oncology therapies.