Sleeping beauty generated CD19 CAR T-Cell therapy for advanced B-Cell hematological malignancies.

Chimeric antigen receptor (CAR) T-cell therapy has emerged recently as a standard of care treatment for patients with relapsed or refractory acute lymphoblastic leukemia (ALL) and several subtypes of B-cell non-Hodgkin lymphoma (NHL). However, its use remains limited to highly specialized centers, given the complexity of its administration and its associated toxicities. We previously reported our experience in using a novel Sleeping Beauty (SB) CD19-specific CAR T-cell therapy in the peri-transplant setting, where it exhibited an excellent safety profile with encouraging survival outcomes. We have since modified the SB CD19 CAR construct to improve its efficacy and shorten its manufacturing time. We report here the phase 1 clinical trial safety results. Fourteen heavily treated patients with relapsed/refractory ALL and NHL were infused. Overall, no serious adverse events were directly attributed to the study treatment. Three patients developed grades 1-2 cytokine release syndrome and none of the study patients experienced neurotoxicity. All dose levels were well tolerated and no dose-limiting toxicities were reported. For efficacy, 3 of 8 (38%) patients with ALL achieved CR/CRi (complete remission with incomplete count recovery) and 1 (13%) patient had sustained molecular disease positivity. Of the 4 patients with DLBCL, 2 (50%) achieved CR. The SB-based CAR constructs allow manufacturing of targeted CAR T-cell therapies that are safe, cost-effective and with encouraging antitumor activity.

Targeting PP2A activates AMPK signaling to inhibit colorectal cancer cells.

LB-100 is a novel PP2A inhibitor. Its activity in human colorectal cancer (CRC) cells was tested. The in vitro studies demonstrated that LB-100 inhibited survival and proliferation of both established CRC cells (HCT-116 and HT-29 lines) and primary human colon cancer cells. Further, LB-100 activated apoptosis and induced G1-S cell cycle arrest in CRC cells. LB-100 inhibited PP2A activity and activated AMPK signaling in CRC cells. AMPKα1 dominant negative mutation, shRNA-mediated knockdown or complete knockout (by CRISPR/Cas9 method) largely attenuated LB-100-induced AMPK activation and HCT-116 cytotoxicity. Notably, microRNA-17-92-mediated silence of PP2A (regulatory B subunit) also activated AMPK and induced HCT-116 cell death. Such effects were again largely attenuated by AMPKα mutation, silence or complete knockout. In vivo studies showed that intraperitoneal injection of LB-100 inhibited HCT-116 xenograft growth in nude mice. Its anti-tumor activity was largely compromised against HCT-116 tumors-derived from AMPKα1-knockout cells. We conclude that targeting PP2A by LB-100 and microRNA-17-92 activates AMPK signaling to inhibit CRC cells.

Connexin45 regulates endothelial-induced mesenchymal cell differentiation toward a mural cell phenotype.

OBJECTIVE: The focus of this study was to investigate the role of connexin (Cx) 45 in endothelial-induced mural cell differentiation. METHODS AND RESULTS: We created mural cell precursors that stably express only Cx45 in Cx43-deficient mesenchymal cells (ReCx45), and used our in vitro model of blood vessel assembly to assess the capacity of this Cx to support endothelial-induced mural cell differentiation. Lucifer Yellow dye injection and dual whole-cell patch clamping revealed that functional gap junctions exhibiting properties of Cx45-containing channels formed among ReCx45 transfectants, and between ReCx45 and endothelial cells. Heterocellular Cx45-containing gap junction channels enabled transforming growth factor-ß activation and promoted the upregulation of mural cell-specific proteins in the mesenchymal precursors. CONCLUSIONS: These studies reveal a critical role for Cx45 in the regulation of endothelial-induced mural cell differentiation, which is consistent with the phenotype of Cx45-deficient embryos that exhibit dysregulated transforming growth factor-ß and lack mural cell development.

KATP channel openers facilitate glutamate uptake by GluTs in rat primary cultured astrocytes.

Increasing evidence, including from our laboratory, has revealed that opening of ATP sensitive potassium channels (KATP channels) plays the neuronal protective roles both in vivo and in vitro. Thus KATP channel openers (KCOs) have been proposed as potential neuroprotectants. Our previous studies demonstrated that KATP channels could regulate glutamate uptake activity in PC12 cells as well as in synaptosomes of rats. Since glutamate transporters (GluTs) of astrocytes play crucial roles in glutamate uptake and KATP channels are also expressed in astrocytes, the present study showed whether and how KATP channels regulated the function of GluTs in primary cultured astrocytes. The results showed that nonselective KCO pinacidil, selective mitochondrial KCO diazoxide, novel, and blood-brain barrier permeable KCO iptakalim could enhance glutamate uptake, except for the sarcolemmal KCO P1075. Moreover pinacidil, diazoxide, and iptakalim reversed the inhibition of glutamate uptake induced by 1-methyl-4-phenylpyridinium (MPP+). These potentiated effects were completely abolished by mitochondrial KATP blocker 5-hydroxydecanoate. Furthermore, either diazoxide or iptakalim could inhibit MPP+-induced elevation of reactive oxygen species (ROS) and phosphorylation of protein kinases C (PKC). These findings are the first to demonstrate that activation of KATP channel, especially mitochondrial KATP channel, improves the function of GluTs in astrocytes due to reducing ROS production and downregulating PKC phosphorylation. Therefore, the present study not only reveals a novel pharmacological profile of KCOs as regulators of GluTs, but also provides a new strategy for neuroprotection.

Ouabain- and marinobufagenin-induced proliferation of human umbilical vein smooth muscle cells and a rat vascular smooth muscle cell line, A7r5.

BACKGROUND: We studied the growth-promoting effects of 2 sodium pump-selective cardiotonic steroids, ouabain and marinobufagenin, on cultured cells from vascular smooth muscle (VSMCs) from human umbilical vein and a rat VSMC line, A7r5. METHODS AND RESULTS: Both ouabain and marinobufagenin activated proliferation of these cells in a concentration-dependent manner, reflecting the cardiotonic steroid sensitivity of the specific alpha1 subunit contained within each cell source. The observed effective concentration ranges of both compounds was below that necessary to induce cytoplasmic ion alterations by sodium pump inhibition. CONCLUSIONS: These data indicate that the ouabain-activated proliferative effect previously observed in canine VSMCs occurs in other VSMC sources. This growth effect seems to be initiated by drug interaction with the sodium pump, reflected by the affinity of the steroid for the pump, and is independent of altered transmembrane ionic gradients.

Gap junction communication mediates transforming growth factor-beta activation and endothelial-induced mural cell differentiation.

During blood vessel assembly, endothelial cells recruit mesenchymal progenitors and induce their differentiation into mural cells via contact-dependent transforming growth factor-beta (TGF-beta) activation. We investigated whether gap junction channels are formed between endothelial cells and recruited mesenchymal progenitors and whether intercellular communication is necessary for endothelial-induced mural cell differentiation. Mesenchymal progenitors from Cx43-/- murine embryos and Cx43+/+ littermates were cocultured with prelabeled endothelial cells. Intracellular dye injection and dual whole-cell voltage clamp revealed that endothelial cells formed gap junction channels with Cx43+/+ but not Cx43-/- progenitors. In coculture with endothelial cells, Cx43-/- progenitors did not undergo mural cell differentiation as did Cx43+/+ cells. Stable reexpression of Cx43 in Cx43-/- cells (reCx43) restored their ability to form gap junctions with endothelial cells and undergo endothelial-induced mural cell differentiation. Cocultures of endothelial cells and either Cx43+/+ or reCx43 mesenchymal cells produced activated TGF-beta; endothelial-Cx43-/- cocultures did not. However, Cx43-/- cells did produce latent TGF-beta and undergo mural cell differentiation in response to exogenous TGF-beta1. These studies indicate that gap junction communication between endothelial and mesenchymal cells mediates TGF-beta activation and subsequent mural cell differentiation.