New Mechanism for Release of Endosomal Contents: Osmotic Lysis via Nigericin-Mediated K+/H+ Exchange.

Although peptides, antibodies/antibody fragments, siRNAs, antisense DNAs, enzymes, and aptamers are all under development as possible therapeutic agents, the breadth of their applications has been severely compromised by their inability to reach intracellular targets. Thus, while macromolecules can often enter cells by receptor-mediated endocytosis, their missions frequently fail due to an inability to escape their entrapping endosomes. In this paper, we describe a general method for promoting release of any biologic material from any entrapping endosome. The strategy relies on the fact that all nascent endosomes contain extracellular (Na+-enriched) medium, but are surrounded by intracellular (K+-enriched) fluid in the cytoplasm. Osmotic swelling and rupture of endosomes will therefore be facilitated if the flow of K+ down its concentration gradient from the cytosol into the endosome can be facilitated without allowing downhill flow of Na+ from the endosome into the cytosol. While any K+ selective ionophore can promote the K+ specific influx, the ideal K+ ionophore will also exchange influxed K+ for an osmotically inactive proton (H+) in order to prevent buildup of an electrical potential that would rapidly halt K+ influx. The only ionophore that catalyzes this exchange of K+ for H+ efficiently is nigericin. We demonstrate here that ligand-targeted delivery of nigericin into endosomes that contain an otherwise impermeable fluorescent dye can augment release of the dye into the cell cytosol via swelling/bursting of the entrapping endosomes. We further show that nigericin-facilitated escape of a folate-targeted luciferase siRNA conjugate from its entrapping endosomes promotes rapid suppression of the intended luciferase reporter gene. Taken together, we propose that ionophore-catalyzed entry of K+ into endosomal compartments can promote the release of otherwise impermeable contents from their encapsulating endosomes.

Therapy-related acute lymphoblastic leukemia has distinct clinical and cytogenetic features compared to de novo acute lymphoblastic leukemia, but outcomes are comparable in transplanted patients.

Therapy-related acute lymphoblastic leukemia remains poorly defined due to a lack of large data sets recognizing the defining characteristics of this entity. We reviewed all consecutive cases of adult acute lymphoblastic leukemia treated at our institution between 2000 and 2017 and identified therapy-related cases - defined as acute lymphoblastic leukemia preceded by prior exposure to cytotoxic chemotherapy and/or radiation. Of 1022 patients with acute lymphoblastic leukemia, 93 (9.1%) were classified as therapy-related. The median latency for therapy-related acute lymphoblastic leukemia onset was 6.8 years from original diagnosis, and this was shorter for patients carrying the MLL gene rearrangement compared to those with other cytogenetics. When compared to de novo acute lymphoblastic leukemia, therapy-related patients were older (P<0.01), more often female (P<0.01), and had more MLL gene rearrangement (P<0.0001) and chromosomes 5/7 aberrations (P=0.02). Although therapy-related acute lymphoblastic leukemia was associated with inferior 2-year overall survival compared to de novo cases (46.0% vs 68.1%, P=0.001), prior exposure to cytotoxic therapy (therapy-related) did not independently impact survival in multivariate analysis (HR=1.32; 95% CI: 0.97-1.80, P=0.08). There was no survival difference (2-year = 53.4% vs 58.9%, P=0.68) between the two groups in patients who received allogenic hematopoietic cell transplantation. In conclusion, therapy-related acute lymphoblastic leukemia represents a significant proportion of adult acute lymphoblastic leukemia diagnoses, and a subset of cases carry clinical and cytogenetic abnormalities similar to therapy-related myeloid neoplasms. Although survival of therapy-related acute lymphoblastic leukemia was inferior to de novo cases, allogeneic hematopoietic cell transplantation outcomes were comparable for the two entities.

Effect of receptor occupancy on folate receptor internalization.

The folate receptor (FR) is a GPI anchored cell surface glycoprotein that functions to facilitate folic acid uptake and mediate signal transduction. With the introduction of multiple folate-targeted drugs into the clinic, the question has arisen regarding how frequently a patient can be dosed with a FR-targeted drug or antibody and whether dosing frequency exerts any impact on the availability of FR for subsequent rounds of FR-mediated drug uptake. Although the rate of FR recycling has been examined in murine tumor models, little or no information exists on the impact of FR occupancy on its rate of endocytosis. The present study quantitates the number of cell surface FR-α and FR-ß following exposure to saturating concentrations of a variety of folate-linked molecules and anti-FR antibodies, including the unmodified vitamin, folate-linked drug mimetics, multifolate derivatized nanoparticles, and monoclonal antibodies to FR. We report here that FR occupancy has no impact on the rate of FR internalization. We also demonstrate that multivalent conjugates that bind and cross-link FRs at the cell surface internalize at the same rate as monovalent folate conjugates that have no impact on FR clustering, even though the multivalent conjugates traffic through a different endocytic pathway.

The Cerebroventricular Environment Modifies CAR T Cells for Potent Activity against Both Central Nervous System and Systemic Lymphoma.

Lymphomas with central nervous system (CNS) involvement confer a worse prognosis than those without CNS involvement, and patients currently have limited treatment options. T cells genetically engineered with CD19-targeted chimeric antigen receptors (CAR) are effective against B-cell malignancies and show tremendous potential in the treatment of systemic lymphoma. We aimed to leverage this strategy toward a more effective therapy for patients with lymphoma with CNS disease. NOD-scid IL2Rgammanull (NSG) mice with CNS and/or systemic lymphoma were treated with CD19-CAR T cells via intracerebroventricular (ICV) or intravenous (IV) injection. CAR T cells isolated after treatment were rigorously examined for phenotype, gene expression, and function. We observed that CAR T cells infused ICV, but not IV, completely and durably eradicated both CNS and systemic lymphoma. CAR T cells delivered ICV migrated efficiently to the periphery, homed to systemic tumors, and expanded in vivo, leading to complete elimination of disease and resistance to tumor rechallenge. Mechanistic studies indicated that ICV-delivered CAR T cells are conditioned by exposure to cerebrospinal fluid in the ICV environment for superior antilymphoma activity and memory function compared with IV-delivered CAR T cells. Further analysis suggested that manipulating cellular metabolism or preactivating therapeutic CAR T cells with antigen ex vivo may improve the efficacy of CAR T cells in vivo Our demonstration that ICV-delivered CD19-CAR T cells had activity against CNS and systemic lymphoma could offer a valuable new strategy for treatment of B-cell malignancies with CNS involvement.

Folate-Hapten-Mediated Immunotherapy Synergizes with Vascular Endothelial Growth Factor Receptor Inhibitors in Treating Murine Models of Cancer.

The overexpression of folate receptors (FR) on many human cancers has led to the development of folate-linked drugs for the imaging and therapy of FR-expressing cancers. In a recent phase I clinical trial of late-stage renal cell carcinoma patients, folate was exploited to deliver an immunogenic hapten, fluorescein, to FR+ tumor cells in an effort to render the cancer cells more immunogenic. Although >50% of the patients showed prolonged stable disease, all patients eventually progressed, suggesting that the folate-hapten immunotherapy was insufficient by itself to treat the cancer. In an effort to identify a companion therapy that might augment the folate-hapten immunotherapy, we explored coadministration of two approved cancer drugs that had been previously shown to also stimulate the immune system. We report that sunitinib and axitinib (VEGF receptor inhibitors that simultaneously mitigate immune suppression) synergize with the folate-hapten-targeted immunotherapy to reduce tumor growth in three different syngeneic murine tumor models. We further demonstrate that the combination therapy not only enhances tumor infiltration of CD4+ and CD8+ effector cells, but surprisingly reduces tumor neovasculogenesis more than predicted. Subsequent investigation of the mechanism for this unexpected suppression of neovasculogenesis revealed that it is independent of elimination of any tumor cells, but instead likely derives from a reduction in the numbers of FR+ tumor-associated macrophages and myeloid-derived suppressor cells, that is, immunosuppressive cells that release significant quantities of VEGF. These data suggest that a reduction in stromal cells of myeloid origin can inhibit tumor growth by suppressing neovasculogenesis. Mol Cancer Ther; 16(3); 461-8. ©2016 AACR.