ABSTRACT
Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP2 binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera.
Subject(s)
Hematopoiesis/genetics , Monomeric Clathrin Assembly Proteins/genetics , Polycythemia Vera/genetics , Anemia, Hypochromic/genetics , Animals , Cell Differentiation , Clathrin-Coated Vesicles/metabolism , Clathrin-Coated Vesicles/ultrastructure , Disease Models, Animal , Endocytosis , Erythroblasts/metabolism , Erythroblasts/ultrastructure , Erythropoiesis/genetics , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Immunophenotyping , Lymphopoiesis/genetics , Mice , Mice, Knockout , Monomeric Clathrin Assembly Proteins/chemistry , Monomeric Clathrin Assembly Proteins/deficiency , Monomeric Clathrin Assembly Proteins/metabolism , Myelopoiesis/genetics , Phenotype , Phosphatidylinositol 4,5-Diphosphate/metabolism , Polycythemia Vera/mortality , Protein Interaction Domains and Motifs , Receptors, Transferrin/metabolismABSTRACT
PURPOSE: Small cell lung cancer (SCLC) is an aggressive disease with limited treatment options. Delta-like ligand 3 (DLL3) is highly expressed on SCLC and several other types of neuroendocrine cancers, with limited normal tissue RNA expression in brain, pituitary, and testis, making it a promising CAR T-cell target for SCLC and other solid tumor indications. EXPERIMENTAL DESIGN: A large panel of anti-DLL3 scFv-based CARs were characterized for both in vitro and in vivo activity. To understand the potential for pituitary and brain toxicity, subcutaneous or intracranial tumors expressing DLL3 were implanted in mice and treated with mouse cross-reactive DLL3 CAR T cells. RESULTS: A subset of CARs demonstrated high sensitivity for targets with low DLL3 density and long-term killing potential in vitro. Infusion of DLL3 CAR T cells led to robust antitumor efficacy, including complete responses, in subcutaneous and systemic SCLC in vivo models. CAR T-cell infiltration into intermediate and posterior pituitary was detected, but no tissue damage in brain or pituitary was observed, and the hormone-secretion function of the pituitary was not ablated. CONCLUSIONS: In summary, the preclinical efficacy and safety data presented here support further evaluation of DLL3 CAR T cells as potential clinical candidates for the treatment of SCLC.
Subject(s)
Hematopoietic Stem Cell Transplantation , Lung Neoplasms , Small Cell Lung Carcinoma , Animals , Male , Mice , Ligands , Lung Neoplasms/drug therapy , Small Cell Lung Carcinoma/genetics , T-Lymphocytes/metabolismABSTRACT
CD70 is highly expressed in renal cell carcinoma (RCC), with limited expression in normal tissue, making it an attractive CAR T target for an immunogenic solid tumor indication. Here we generated and characterized a panel of anti-CD70 single-chain fragment variable (scFv)-based CAR T cells. Despite the expression of CD70 on T cells, production of CAR T cells from a subset of scFvs with potent in vitro activity was achieved. Expression of CD70 CARs masked CD70 detection in cis and provided protection from CD70 CAR T cell-mediated fratricide. Two distinct classes of CAR T cells were identified with differing memory phenotype, activation status, and cytotoxic activity. Epitope mapping revealed that the two classes of CARs bind unique regions of CD70. CD70 CAR T cells displayed robust antitumor activity against RCC cell lines and patient-derived xenograft mouse models. Tissue cross-reactivity studies identified membrane staining in lymphocytes, thus matching the known expression pattern of CD70. In a cynomolgus monkey CD3-CD70 bispecific toxicity study, expected findings related to T-cell activation and elimination of CD70-expressing cells were observed, including cytokine release and loss of cellularity in lymphoid tissues. Finally, highly functional CD70 allogeneic CAR T cells were produced at large scale through elimination of the T-cell receptor by TALEN-based gene editing. Taken together, these efficacy and safety data support the evaluation of CD70 CAR T cells for the treatment of RCC and has led to the advancement of an allogeneic CD70 CAR T-cell candidate into phase I clinical trials. SIGNIFICANCE: These findings demonstrate the efficacy and safety of fratricide-resistant, allogeneic anti-CD70 CAR T cells targeting renal cell carcinoma and the impact of CAR epitope on functional activity. See related commentary by Adotévi and Galaine, p. 2517.
Subject(s)
Carcinoma, Renal Cell , Hematopoietic Stem Cell Transplantation , Kidney Neoplasms , Animals , CD27 Ligand , Carcinoma, Renal Cell/pathology , Cell Line, Tumor , Humans , Immunotherapy, Adoptive , Kidney Neoplasms/pathology , Macaca fascicularis , Mice , T-Lymphocytes/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Medulloblastoma is among the most common malignant brain tumors in children. Recent studies have identified at least four subgroups of the disease that differ in terms of molecular characteristics and patient outcomes. Despite this heterogeneity, most patients with medulloblastoma receive similar therapies, including surgery, radiation, and intensive chemotherapy. Although these treatments prolong survival, many patients still die from the disease and survivors suffer severe long-term side effects from therapy. We hypothesize that each patient with medulloblastoma is sensitive to different therapies and that tailoring therapy based on the molecular and cellular characteristics of patients' tumors will improve outcomes. To test this, we assembled a panel of orthotopic patient-derived xenografts (PDX) and subjected them to DNA sequencing, gene expression profiling, and high-throughput drug screening. Analysis of DNA sequencing revealed that most medulloblastomas do not have actionable mutations that point to effective therapies. In contrast, gene expression and drug response data provided valuable information about potential therapies for every tumor. For example, drug screening demonstrated that actinomycin D, which is used for treatment of sarcoma but rarely for medulloblastoma, was active against PDXs representing Group 3 medulloblastoma, the most aggressive form of the disease. Functional analysis of tumor cells was successfully used in a clinical setting to identify more treatment options than sequencing alone. These studies suggest that it should be possible to move away from a one-size-fits-all approach and begin to treat each patient with therapies that are effective against their specific tumor. SIGNIFICANCE: These findings show that high-throughput drug screening identifies therapies for medulloblastoma that cannot be predicted by genomic or transcriptomic analysis.
Subject(s)
Antineoplastic Agents/pharmacology , Cerebellar Neoplasms/drug therapy , Medulloblastoma/drug therapy , Precision Medicine/methods , Animals , Cell Line, Tumor , Cerebellar Neoplasms/genetics , Child , Dactinomycin/pharmacology , Gene Expression Regulation, Neoplastic , High-Throughput Screening Assays , Humans , Male , Medulloblastoma/genetics , Mice, Inbred NOD , Mutation , Polymorphism, Single Nucleotide , Exome Sequencing , Xenograft Model Antitumor AssaysABSTRACT
Drugs that modify the epigenome are powerful tools for treating cancer, but these drugs often have pleiotropic effects, and identifying patients who will benefit from them remains a major clinical challenge. Here we show that medulloblastomas driven by the transcription factor Gfi1 are exquisitely dependent on the enzyme lysine demethylase 1 (Kdm1a/Lsd1). We demonstrate that Lsd1 physically associates with Gfi1, and that these proteins cooperate to inhibit genes involved in neuronal commitment and differentiation. We also show that Lsd1 is essential for Gfi1-mediated transformation: Gfi1 proteins that cannot recruit Lsd1 are unable to drive tumorigenesis, and genetic ablation of Lsd1 markedly impairs tumor growth in vivo. Finally, pharmacological inhibitors of Lsd1 potently inhibit growth of Gfi1-driven tumors. These studies provide important insight into the mechanisms by which Gfi1 contributes to tumorigenesis, and identify Lsd1 inhibitors as promising therapeutic agents for Gfi1-driven medulloblastoma.
Subject(s)
Carcinogenesis/drug effects , Cerebellar Neoplasms/pathology , DNA-Binding Proteins/metabolism , Histone Demethylases/metabolism , Medulloblastoma/pathology , Transcription Factors/metabolism , Animals , Antibiotics, Antineoplastic/therapeutic use , Cell Proliferation/drug effects , Cerebellar Neoplasms/genetics , Cerebellar Neoplasms/therapy , DNA-Binding Proteins/genetics , Doxorubicin/therapeutic use , Gene Expression Regulation, Neoplastic , HEK293 Cells , Histone Demethylases/genetics , Humans , Medulloblastoma/genetics , Medulloblastoma/therapy , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, SCID , NIH 3T3 Cells , Neoplasm Transplantation , Oncogenic Viruses , Retroviridae , Transcription Factors/geneticsABSTRACT
Itraconazole (ITZ) is an FDA-approved member of the triazole class of antifungal agents. Two recent drug repurposing screens identified ITZ as a promising anticancer chemotherapeutic that inhibits both the angiogenesis and hedgehog (Hh) signaling pathways. We have synthesized and evaluated first- and second-generation ITZ analogues for their anti-Hh and antiangiogenic activities to probe more fully the structural requirements for these anticancer properties. Our overall results suggest that the triazole functionality is required for ITZ-mediated inhibition of angiogenesis but that it is not essential for inhibition of Hh signaling. The synthesis and evaluation of stereochemically defined des-triazole ITZ analogues also provides key information as to the optimal configuration around the dioxolane ring of the ITZ scaffold. Finally, the results from our studies suggest that two distinct cellular mechanisms of action govern the anticancer properties of the ITZ scaffold.
Subject(s)
Antifungal Agents/therapeutic use , Antineoplastic Agents/therapeutic use , Itraconazole/therapeutic use , Animals , Cell Line , Inhibitory Concentration 50 , Mice , Mice, Inbred C3H , RNA, Messenger/genetics , Zinc Finger Protein GLI1/geneticsABSTRACT
How does mitosis influence the critical process of endocytosis? Some experiments lead to the conclusion that endocytosis arrests completely during mitosis, whereas others indicate that endocytosis persists. We have resolved this apparent discrepancy by showing how conditions of the experiment influence its outcome. The dynamics of clathrin-coated pit formation and the uptake of transferrin are maintained in naturally dividing cells but are nearly absent in mitotic cells arrested chemically by treatment with nocodazole, S-Trityl-L-cysteine, or RO-3306. Moreover, sequentially incubating cells at 4°C and then shifting them to 37°C or to serum starvation artificially increases the amount of transferrin receptor at the surface of naturally dividing cells, leading to the incorrect conclusion that endocytosis has ceased during mitosis. Thus, our data show that endocytosis is unaffected during all stages of natural cell division.
Subject(s)
Clathrin/metabolism , Endocytosis , Mitosis/physiology , Animals , Chlorocebus aethiops , Clathrin-Coated Vesicles/metabolism , Cysteine/analogs & derivatives , Cysteine/pharmacology , HEK293 Cells , HeLa Cells , Hot Temperature , Humans , Mitosis/drug effects , Nocodazole/pharmacology , Quinolines/pharmacology , Thiazoles/pharmacology , Transferrin/metabolismABSTRACT
Clathrin/AP1- and clathrin/AP3-coated vesicular carriers originate from endosomes and the trans-Golgi network. Here, we report the real-time visualization of these structures in living cells reliably tracked by rapid, three-dimensional imaging with the use of a spinning-disk confocal microscope. We imaged relatively sparse, diffraction-limited, fluorescent objects containing chimeric fluorescent protein (clathrin light chain, σ adaptor subunits, or dynamin2) with a spatial precision of up to ~30 nm and a temporal resolution of ~1 s. The dynamic characteristics of the intracellular clathrin/AP1 and clathrin/AP3 carriers are similar to those of endocytic clathrin/AP2 pits and vesicles; the clathrin/AP1 coats are, on average, slightly shorter-lived than their AP2 and AP3 counterparts. We confirmed that although dynamin2 is recruited as a burst to clathrin/AP2 pits immediately before their budding from the plasma membrane, we found no evidence supporting a similar association of dynamin2 with clathrin/AP1 or clathrin/AP3 carriers at any stage during their lifetime. We found no effects of chemical inhibitors of dynamin function or the K44A dominant-negative mutant of dynamin on AP1 and AP3 dynamics. This observation suggests that an alternative budding mechanism, yet to be discovered, is responsible for the scission step of clathrin/AP1 and clathrin/AP3 carriers.