Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD leukemia cells. This synergized with the allogeneic CD8 T cell response, leading to long-term survival in six mouse models of FLT3-ITD AML. Sorafenib-related IL-15 production caused an increase in CD8CD107aIFN-γ T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8 T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.
A chemical investigation of the endophytic fungus Epicoccum nigrum isolated from leaves of Mentha suaveolens collected in Morocco resulted in the isolation of five new polyketides, epicocconigrones A and B (1 and 2), 3-methoxyepicoccone B (3), 3-methoxyepicoccone (4), and 2,3,4-trihydroxy-6-(methoxymethyl)-5-methylbenzaldehyde (5), together with five known compounds (6-10). The structures of the new compounds were unambiguously determined by extensive analysis of the 1D and 2D NMR and mass spectroscopic data. Compounds 1 and 10 showed potent inhibition of at least 15 protein kinases with IC50 values ranging from 0.07 to 9.00 μM. Moreover, compounds 1 and 10 inhibited histone deacetylase (HDAC) activities with IC50 values of 9.8 and 14.2 μM, respectively. A preliminary structure-activity relationship is discussed. Interestingly, compounds 1 and 10 exert mainly cytostatic effects in human lymphoma RAJI and U-937 cell lines.
Chemical investigation of the sponge Dactylospongia metachromia afforded five new sesquiterpene aminoquinones (1-5), two new sesquiterpene benzoxazoles (6 and 7), the known analogue 18-hydroxy-5-epi-hyrtiophenol (8), and a known glycerolipid. The structures of all compounds were unambiguously elucidated by one- and two-dimensional NMR and by MS analyses, as well as by comparison with the literature. Compounds 1-5 showed potent cytotoxicity against the mouse lymphoma cell line L5178Y with IC50 values ranging from 1.1 to 3.7 μM. When tested in vitro for their inhibitory potential against 16 different protein kinases, compounds 5, 6, and 8 exhibited the strongest inhibitory activity against ALK, FAK, IGF1-R, SRC, VEGF-R2, Aurora-B, MET wt, and NEK6 kinases (IC50 0.97-8.62 μM).
Technical liquid flow films are the basic arrangement for gas fluid transitions of all kinds and are the basis of many chemical processes, such as columns, evaporators, dryers, and different other kinds of fluid/fluid separation units. This publication presents a new method for molecule sensitive, non-contact, and marker-free localized concentration mapping in vertical falling films. Using Raman spectroscopy, no label or marker is needed for the detection of the local composition in liquid mixtures. In the presented cases, the film mapping of sodium sulfate in water on a plain surface as well as an added artificial streaming disruptor with the shape of a small pyramid is scanned in three dimensions. The results show, as a prove of concept, a clear detectable spectroscopic difference between air, back plate, and sodium sulfate for every local point in all three dimensions. In conclusion, contactless Raman scanning on falling films for liquid mapping is realizable without any mechanical film interaction caused by the measuring probe. Surface gloss or optical reflections from a metallic back plate are suppressed by using only inelastic light scattering and the mathematical removal of background noise.
Bispecific antibodies are being explored as a means to modulate responses of the immune system to tumor infiltration. They are emerging as a growing class of immunotherapies with potential to further improve clinical efficacy and safety. Besides tumor-targeted and dual immunomodulators, the most important class of therapeutically relevant bispecific antibodies are engagers of cytotoxic immune cells to kill tumor cells. These are mainly engager of T-cells (BiTE´s), but also NK-cell engager several of which have entered clinics with Blinatumomab (anti-CD3/anti-CD19) being FDA-approved for the treatment of Acute Lymphoblastic Leukemia. Several studies are under way to address potential combinatorial effects with other compounds in clinics. In order to facilitate analysis of the cytotoxic impact of BiTE´s in high throughput, we developed a 384-well format assay system based on Luciferase-labeled tumor cells. This detection technology has many advantages. As only the tumor cells are labeled, the specific detection of luciferase exclusively correlates to the number of viable tumor cells, allowing for co-cultures with high excess of effector cells without them interfering in the detection. Due to high sensitivity, only few tumor cells are required for a decent signal/noise ratio, keeping the overall need for primary effector cells to the lowest, even at high effector/target cell ratios. That can be of great advantage when working with rare cytotoxic subpopulations (e.g. NK-cells), precious samples from patients, or simply saves resources when performing large exploratory studies. In addition, combination of low 384-well format volumes with nanodrop agent dispensing technology minimizes amounts of expensive antibodies and other agents. Applying this technology, we evaluated the combinatorial effects of Blinatumomab with immune modulators (e.g. Lenalidomide), kinase inhibitors (e.g. MEK-inhibitor, Selumetinib) and other clinically relevant agents (e.g. MCL1-inhibitor, S63845) on the Luciferase-labeled GCB-like DLBCL cell line OCI-LY1. Depending on compound combination, we observe synergistic but also antagonistic effects. Our data support the outstanding usefulness of this methodological approach for the exploration of bispecific cytotoxic immune cell engager agents.
Citation Format: Carla N. Castro, Daniel Feger, Sarah Ulrich, Oliver Siedentopf, Jan E. Ehlert. Exploring the combinatorial potential of bispecific T-cell engagers in high throughput format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2893.
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