Extrinsic signaling cues in the microenvironment of acute myelogenous leukemia (AML) contribute to disease progression and therapy resistance. Yet, it remains unknown how the bone marrow niche in which AML arises is subverted to support leukemic persistence at the expense of homeostatic function. Exosomes are cell membrane-derived vesicles carrying protein and RNA cargoes that have emerged as mediators of cell-cell communication. In this study, we examined the role of exosomes in developing the AML niche of the bone marrow microenvironment, investigating their biogenesis with a focus on RNA trafficking. We found that both primary AML and AML cell lines released exosome-sized vesicles that entered bystander cells. These exosomes were enriched for several coding and noncoding RNAs relevant to AML pathogenesis. Furthermore, their uptake by bone marrow stromal cells altered their secretion of growth factors. Proof-ofconcept studies provided additional evidence for the canonical functions of the transferred RNA. Taken together, our findings revealed that AML exosome trafficking alters the proliferative, angiogenic, and migratory responses of cocultured stromal and hematopoietic progenitor cell lines, helping explain how the microenvironmental niche becomes reprogrammed during invasion of the bone marrow by AML. Cancer Res; 73(2); 918-29. Ó2012 AACR.
On May 8, 2020, the FDA granted accelerated approval to selpercatinib for (i) adult patients with metastatic RET fusion–positive non–small cell lung cancer (NSCLC), (ii) adult and pediatric patients ≥12 years of age with advanced or metastatic RET-mutant medullary thyroid cancer who require systemic therapy, and (iii) adult and pediatric patients ≥12 years of age with advanced or metastatic RET fusion–positive thyroid cancer who require systemic therapy and who are radioactive iodine refractory (if radioactive iodine is appropriate). Approval was granted on the basis of the clinically important effects on the overall response rate (ORR) with prolonged duration of responses observed in a multicenter, open-label, multicohort clinical trial (LIBRETTO-001, NCT03157128) in patients whose tumors had RET alterations. ORRs within the approved patient populations ranged from 64% [95% confidence interval (CI), 54–73] in prior platinum-treated RET fusion–positive NSCLC to 100% (95% CI, 63–100) in systemic therapy–naïve RET fusion–positive thyroid cancer, with the majority of responders across indications demonstrating responses of at least 6 months. The product label includes warnings and precautions for hepatotoxicity, hypertension, QT interval prolongation, hemorrhagic events, hypersensitivity, risk of impaired wound healing, and embryo-fetal toxicity. This is the first approval of a drug specifically for patients with RET alterations globally.
The FDA approved capmatinib and tepotinib on May 6, 2020, and February 3, 2021, respectively. Capmatinib is indicated for patients with metastatic non–small cell lung cancer (mNSCLC) whose tumors have a mutation leading to mesenchymal–epithelial transition (MET) exon 14 skipping as detected by an FDA-approved test. Tepotinib is indicated for mNSCLC harboring MET exon 14 skipping alterations. The approvals were based on trials GEOMETRY mono-1 (capmatinib) and VISION (tepotinib). In GEOMETRY mono-1, overall response rate (ORR) per Blinded Independent Review Committee (BIRC) was 68% [95% confidence interval (CI), 48–84] with median duration of response (DoR) 12.6 months (95% CI, 5.5–25.3) in 28 treatment-naïve patients and 41% (95% CI: 29, 53) with median DoR 9.7 months (95% CI, 5.5–13) in 69 previously treated patients with NSCLC with mutations leading to MET exon 14 skipping. In VISION, ORR per BIRC was 43% (95% CI: 32, 56) with median DoR 10.8 months (95% CI, 6.9–not estimable) in 69 treatment-naïve patients and 43% (95% CI, 33–55) with median DoR 11.1 months (95% CI, 9.5–18.5) in 83 previously-treated patients with NSCLC harboring MET exon 14 alterations. These are the first two therapies to be FDA approved specifically for patients with metastatic NSCLC with MET exon 14 skipping.
Insertional oncogene activation and aberrant splicing have proved to be major setbacks for retroviral stem cell gene therapy. Integrase-deficient human immunodeficiency virus-1-derived vectors provide a potentially safer approach, but their circular genomes are rapidly lost during cell division. Here we describe a novel lentiviral vector (LV) that incorporates human ß-interferon scaffold/matrix-associated region sequences to provide an origin of replication for long-term mitotic maintenance of the episomal LTR circles. The resulting ‘anchoring’ non-integrating lentiviral vector (aniLV) achieved initial transduction rates comparable with integrating vector followed by progressive establishment of long-term episomal expression in a subset of cells. Analysis of aniLV-transduced single cell-derived clones maintained without selective pressure for >100 rounds of cell division showed sustained transgene expression from episomes and provided molecular evidence for long-term episome maintenance. To evaluate aniLV performance in primary cells, we transduced lineage-depleted murine hematopoietic progenitor cells, observing GFP expression in clonogenic progenitor colonies and peripheral blood leukocyte chimerism following transplantation into conditioned hosts. In aggregate, our studies suggest that scaffold/matrix-associated region elements can serve as molecular anchors for non-integrating lentivector episomes, providing sustained gene expression through successive rounds of cell division and progenitor differentiation in vitro and in vivo.
SummaryCell fusion plays a well-recognized, physiological role during development. Bone-marrow-derived hematopoietic cells have been shown to fuse with non-hematopoietic cells in a wide variety of tissues. Some organs appear to resolve the changes in ploidy status, generating functional and mitotically-competent events. However, cell fusion exclusively involving hematopoietic cells has not been reported. Indeed, genomic copy number variation in highly replicative hematopoietic cells is widely considered a hallmark of malignant transformation. Here we show that cell fusion occurs between cells of the hematopoietic system under injury as well as non-injury conditions. Experiments reveal the acquisition of genetic markers in fusion products, their tractable maintenance during hematopoietic differentiation and long-term persistence after serial transplantation. Fusion events were identified in clonogenic progenitors as well as differentiated myeloid and lymphoid cells. These observations provide a new experimental model for the study of non-pathogenic somatic diversity in the hematopoietic system.
Background Transduction with recombinant Human Immunodeficiency Virus (HIV) -1 derived lentivirus vectors is a multi-step process initiated by surface attachment and subsequent receptor-directed uptake into the target cell. We previously reported the retention of vesicular stomatitis virus G protein (VSV-G) pseudotyped particles on murine progenitor cells and their delayed cell-cell transfer. Methods To examine the underlying mechanism in more detail we used a combination of approaches focused on investigating the role of receptor-independent factors in modulating attachment. Results Studies of synchronized transduction herein reveal cell-type specific rates of vector particle clearance with substantial delays during particle entry into murine hematopoietic progenitor cells. The observed uptake kinetics from the surface of the 1° cell correlate inversely with the magnitude of transfer to 2° targets, corresponding with our initial observation of preferential cell-cell transfer in the context of brief vector exposures. We further demonstrate that vector particle entry into cells is associated with the cell–type specific abundance of extracellular matrix fibronectin. Residual particle – ECM binding and 2° transfer can be competitively disrupted by heparin exposure without affecting murine progenitor homing and repopulation. Conclusions While cellular attachment factors, including fibronectin, aid gene transfer by colocalizing particles to cells and disfavoring early dissociation from targets, they also appear to stabilize particles on the cell surface. Our study highlights the inadvertent consequences for cell entry and cell-cell transfer.
Uveitis is an inflammatory ocular disease characterized by the infiltration of T lymphocytes and other leukocytes into the eye. The recruitment of these inflammatory cells from systemic vasculature to ocular tissue is a well-coordinated multistep process including rolling, firm adhesion and transmigration. CXCL12 (SDF-1α) is an endothelial cell-derived cytokine interacting with CXCR4 and CXCR7, two chemokine receptors mainly expressed in T cells, neutrophils and monocytes. Recent studies have shown that CXCR4, CXCR7 and their ligand, CXCL12, are important for the regulation of leukocyte mobilization and trafficking. However, it is unclear whether these two chemokine receptors are implicated in the pathogenesis of uveitis. In this study, we used DO11.10 mice, whose CD4+ T cells are genetically engineered to react with ovalbumin (OVA), to investigate the role of CXCR4 and CXCR7 in an animal model of uveitis. Intravital microscopy revealed that intravitreal OVA challenge of DO11.10 mice caused the infiltration of both T cells and neutrophils. The invasion of these inflammatory cells coincided with the detection of transcriptional upregulation of CXCR4 and CXCR7 in the eye. In addition, both real time-PCR and immunohistochemistry revealed an enhanced expression of endothelial CXCL12. Furthermore, intraperitoneal injection of AMD3100 (a specific CXCR4 antagonist) significantly attenuated OVA-induced uveitis and CXCL12-mediated transwell migration. In contrast, intraperitoneal administration of CXCR7 neutralizing antibody did not significantly alter ocular infiltration of inflammatory cells caused by OVA challenge. Our data suggest that CXCR4 but not CXCR7 plays a critical role in antigen-induced ocular inflammation by facilitating leukocyte infiltration. This study not only enhances our knowledge of the immunopathological mechanism of uveitis but also provides a novel rationale to target CXCR4 as an anti-inflammatory strategy to treat uveitis.
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