Uveal melanoma (UM) is the most common intraocular malignant tumor. Despite successful treatment of the primary tumor, about 50% of patients will recur with systemic disease for which there are no ...effective treatment strategies. Here we investigated the preclinical efficacy of a chimeric antigen receptor (CAR) T cell-based immunotherapy targeting B7-H3.
B7-H3 expression on primary and metastatic human UM samples and cell lines was assessed by RNA sequencing, flow cytometry, and immunohistochemistry. Antitumor activity of CAR T cells targeting B7-H3 was tested in vitro with UM cell lines, metastatic UM patient-derived organotypic tumor spheroids (PDOTS), and in immunodeficient and humanized murine models.
B7-H3 is expressed at high levels on >95% UM tumor cells in vitro and in vivo. We generated a B7-H3 CAR with an inducible caspase-9 (iCas9) suicide gene controlled by the chemical inducer of dimerization AP1903, which effectively kills UM cells in vitro and eradicates UM liver metastases in murine models. Delivery of iCas9.B7-H3 CAR T cells in experimental models of UM liver metastases demonstrates a durable anti-tumor response, even upon tumor re-challenge or in the presence of a significant metastatic disease burden. We demonstrate effective iCas9.B7-H3 CAR T cell elimination in vitro and in vivo in response to AP1903. Our studies demonstrate more effective tumor suppression with iCas9.B7-H3 CAR T cells as compared to a B7-H3-targeted humanized monoclonal antibody.
These studies support a phase I clinical trial with iCas9.B7-H3 CAR T cells to treat patients with metastatic UM.
Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking
. Here we identify the innate immune ...kinase TANK-binding kinase 1 (TBK1)
as a candidate immune-evasion gene in a pooled genetic screen
. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK, ZAGLJ
Chronic kidney disease and end-stage renal disease are major causes of morbidity and mortality that are seen far more commonly in the aged population. Interestingly, kidney function declines during ...aging even in the absence of underlying renal disease. Declining renal function has been associated with age-related cellular damage and dysfunction with reports of increased levels of apoptosis, necrosis, and inflammation in the aged kidney. Bioactive sphingolipids have been shown to regulate these same cellular processes, and have also been suggested to play a role in aging and cellular senescence.
We hypothesized that alterations in kidney sphingolipids play a role in the declining kidney function that occurs during aging. To begin to address this, the sphingolipid profile was measured in young (3 mo), middle aged (9 mo) and old (17 mo) C57BL/6 male mice. Interestingly, while modest changes in ceramides and sphingoid bases were evident in kidneys from older mice, the most dramatic elevations were seen in long-chain hexosylceramides (HexCer) and lactosylceramides (LacCer), with C14- and C16-lactosylceramides elevated as much as 8 and 12-fold, respectively. Increases in long-chain LacCers during aging are not exclusive to the kidney, as they also occur in the liver and brain. Importantly, caloric restriction, previously shown to prevent the declining kidney function seen in aging, inhibits accumulation of long-chain HexCer/LacCers and prevents the age-associated elevation of enzymes involved in their synthesis. Additionally, long-chain LacCers are also significantly elevated in human fibroblasts isolated from elderly individuals.
This study demonstrates accumulation of the glycosphingolipids HexCer and LacCer in several different organs in rodents and humans during aging. In addition, data demonstrate that HexCer and LacCer metabolism is regulated by caloric restriction. Taken together, data suggest that HexCer/LacCers are important mediators of cellular processes fundamental to mammalian aging.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed ...cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.
Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment ...(TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells).
Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids.
We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors.
CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.
Analysis of lineage relationships in the naphthalene-injured tracheal epithelium demonstrated that two multipotential keratin 14-expressing cells (K14ECs) function as progenitors for Clara and ...ciliated cells. These K14EC were distinguished by their self-renewal capacity and were hypothesized to reside at the stem and transit amplifying tiers of a tissue-specific stem cell hierarchy. In this study, we used gene expression and histomorphometric analysis of the steady-state and naphthalene-injured trachea to evaluate the predictions of this model. We found that the steady-state tracheal epithelium is maintained by two progenitor cell pools, secretory and basal cells, and the latter progenitor pool is further divided into two subsets, keratin 14-negative and -positive. After naphthalene-mediated depletion of the secretory and ciliated cell types, the two basal cell pools coordinate to restore the epithelium. Both basal cell types up-regulate keratin 14 and generate a broadly distributed, abundant, and highly mitotic cell pool. Furthermore, basal cell proliferation is associated with generation of differentiated Clara and ciliated cells. The uniform distribution of basal cell progenitors and of their differentiated progeny leads us to propose that the hierarchical organization of tracheal reparative cells be revised to include a facultative basal cell progenitor pool.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts ...influence cysteine targeting remains unknown. To address this question, we have developed “DrugMap,” an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.
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•Chemical proteomic analysis of cysteine ligandability across 400+ cancer cell lines•Ligandability is impacted by cellular redox states, protein conformations, and mutations•Covalent ligands disrupt activity of oncogenic transcription factors NF-κB1 and SOX10
DrugMap serves as a roadmap to develop covalent ligands for oncogenic drivers.
The sphingolipid ceramide has been widely implicated in the regulation of programmed cell death or apoptosis. The accumulation of ceramide has been demonstrated in a wide variety of experimental ...models of apoptosis and in response to a myriad of stimuli and cellular stresses. However, the detailed mechanisms of its generation and regulatory role during apoptosis are poorly understood. We sought to determine the regulation and roles of ceramide production in a model of ultraviolet light-C (UV-C)-induced programmed cell death. We found that UV-C irradiation induces the accumulation of multiple sphingolipid species including ceramide, dihydroceramide, sphingomyelin, and hexosylceramide. Late ceramide generation was also found to be regulated by Bcl-xL, Bak, and caspases. Surprisingly, inhibition of de novo synthesis using myriocin or fumonisin B1 resulted in decreased overall cellular ceramide levels basally and in response to UV-C, but only fumonisin B1 inhibited cell death, suggesting the presence of a ceramide synthase (CerS)-dependent, sphingosine-derived pool of ceramide in regulating programmed cell death. We found that this pool did not regulate the mitochondrial pathway, but it did partially regulate activation of caspase-7 and, more importantly, was necessary for late plasma membrane permeabilization. Attempting to identify the CerS responsible for this effect, we found that combined knockdown of CerS5 and CerS6 was able to decrease long-chain ceramide accumulation and plasma membrane permeabilization. These data identify a novel role for CerS and the sphingosine salvage pathway in regulating membrane permeability in the execution phase of programmed cell death.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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