Figure 4.
IL-1β and TNF-α are upregulated in monocytic leukemia and promotes drug resistance to venetoclax and MDM2 inhibitors. (A) The schematic illustrates the coculture drug assay. The leukemia blasts were placed in the upper well with and without idasanutlin or venetoclax. The supporting granulocytes, T cells, and monocytes isolated from leukemia patient samples or healthy donors were cultured in the bottom well. (B) Representative flow cytometry apoptosis assay images of the leukemia blast cells treated with 1 μM idasanutlin in the presence or absence of granulocytes and monocytes isolated from monocytic leukemia samples for 48 hours as determined by annexin V and PI staining. (C) Graphs depict the viabilities of primary leukemia blast cells (22-266) that were cultured with leukemia (L) monocytes, granulocytes, and T cells isolated from sample 22-111 in the presence or absence of idasanutlin (1 μM), determined by annexin V/PI. (D) Graphs depict the viabilities of the primary leukemia blast cells (22-255) that were cultured with L granulocytes and L T cells isolated from 22-111 and L monocytes from 22-111 and 22-255 in the presence or absence of 3 MDM2 inhibitors (1 μM), determined by annexin V/PI. (E) Graphs depict the viabilities of 5 primary leukemia blasts from patients that were cultured with monocytes isolated from an M4 leukemia sample (22-302), in the presence or absence of idasanutlin, as determined by annexin V/PI. (F) Graphs depict the viabilities of blasts from 2 patients with primary leukemia that were cultured with monocytes isolated from 2 M4/M5 leukemia samples in the presence or absence of venetoclax (300 nM) for 2 days, determined using annexin V/PI. (G) Relative expression of the indicated cytokines in 1 million CD14+ cells that were isolated from different primary patient samples and healthy donors for 2 days, determined using a Luminex assay. (H) Graphs depict the normalized viabilities of the indicated FAB cell type that was cultured with either idasanutlin or venetoclax in the presence or absence of several cytokines. Viabilities were normalized to the respective idasanutlin or venetoclax cells. Viabilities differences, in comparison with the drug-only control, were determined using Friedman tests. (I) The dot plots illustrate the positive correlation of interleukin-1 receptor antagonist (IL1RA) and IL-1β with idasanutlin and ILRA, IL-1β, and TNF-α with venetoclax, determined using 2-tailed Pearson correlation tests. (J) The graph depicts the single-cell average RNA expression z-scores of the indicated genes in M4/M5 and M0/M1 leukemia samples. The circle size indicates the percentage of cells that expressed a given gene in the respective cell type. (K) The bar graph depicts the average number of cells in each population from panel J.

IL-1β and TNF-α are upregulated in monocytic leukemia and promotes drug resistance to venetoclax and MDM2 inhibitors. (A) The schematic illustrates the coculture drug assay. The leukemia blasts were placed in the upper well with and without idasanutlin or venetoclax. The supporting granulocytes, T cells, and monocytes isolated from leukemia patient samples or healthy donors were cultured in the bottom well. (B) Representative flow cytometry apoptosis assay images of the leukemia blast cells treated with 1 μM idasanutlin in the presence or absence of granulocytes and monocytes isolated from monocytic leukemia samples for 48 hours as determined by annexin V and PI staining. (C) Graphs depict the viabilities of primary leukemia blast cells (22-266) that were cultured with leukemia (L) monocytes, granulocytes, and T cells isolated from sample 22-111 in the presence or absence of idasanutlin (1 μM), determined by annexin V/PI. (D) Graphs depict the viabilities of the primary leukemia blast cells (22-255) that were cultured with L granulocytes and L T cells isolated from 22-111 and L monocytes from 22-111 and 22-255 in the presence or absence of 3 MDM2 inhibitors (1 μM), determined by annexin V/PI. (E) Graphs depict the viabilities of 5 primary leukemia blasts from patients that were cultured with monocytes isolated from an M4 leukemia sample (22-302), in the presence or absence of idasanutlin, as determined by annexin V/PI. (F) Graphs depict the viabilities of blasts from 2 patients with primary leukemia that were cultured with monocytes isolated from 2 M4/M5 leukemia samples in the presence or absence of venetoclax (300 nM) for 2 days, determined using annexin V/PI. (G) Relative expression of the indicated cytokines in 1 million CD14+ cells that were isolated from different primary patient samples and healthy donors for 2 days, determined using a Luminex assay. (H) Graphs depict the normalized viabilities of the indicated FAB cell type that was cultured with either idasanutlin or venetoclax in the presence or absence of several cytokines. Viabilities were normalized to the respective idasanutlin or venetoclax cells. Viabilities differences, in comparison with the drug-only control, were determined using Friedman tests. (I) The dot plots illustrate the positive correlation of interleukin-1 receptor antagonist (IL1RA) and IL-1β with idasanutlin and ILRA, IL-1β, and TNF-α with venetoclax, determined using 2-tailed Pearson correlation tests. (J) The graph depicts the single-cell average RNA expression z-scores of the indicated genes in M4/M5 and M0/M1 leukemia samples. The circle size indicates the percentage of cells that expressed a given gene in the respective cell type. (K) The bar graph depicts the average number of cells in each population from panel J.

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