MAP3K8 kinase regulates myeloma growth by cell-autonomous and non-autonomous mechanisms involving myeloma-associated monocytes/macrophages

Abstract

Benefit from cytotoxic therapy in myeloma may be limited by the persistence of residual tumour cells within protective niches. We have previously shown that monocytes/macrophages acquire a proinflammatory transcriptional profile in the myeloma microenvironment. Here we report constitutive activation of MAP3K8 kinase-dependent pathways that regulate the magnitude and extent of inflammatory activity of monocytes/macrophages within myeloma niches. In myeloma tumour cells, MAP3K8 acts as mitogen-induced MAP3K in mitosis and is required for TNFα-mediated ERK activation. Pharmacological MAP3K8 inhibition results in dose-dependent, tumour cell-autonomous apoptosis despite contact with primary stroma. MAP3K8 blockade may disrupt crucial macrophage-tumour cell interactions within myeloma niches.

Figure 1

A. MAP3K8 kinase is constitutively active in myeloma-associated monocytes/macrophages. Bone marrow (BM) aspirates were collected with informed consent from 7 patients (“MM” lanes). Fresh BM from normal donors was obtained commercially. CD14+ mononuclear cells were purified by immunomagnetic separation and lysates were immunoblotted using antisera against phosphorylated (phospho-Thr290) and total MAP3K8. For the normal bone marrow control, CD14+ denotes the CD14+ mononuclear cell fraction (positive isolation) and CD14- denotes the CD14-depleted mononuclear fraction. Two different normal marrow donors gave identical results. Both p58 and p52 MAP3K8 isoforms are expressed in CD14+ cells at comparable rates. The phosphorylated p58 isoform is prone to rapid degradation (Gantke, et al 2011). B. Constitutive ERK activation in myeloma-associated monocytes/macrophages. The activation status of ERK was probed using antibodies against phosphorylated and total ERK. CD14+ peripheral blood monocytes from 5 different normal donors are shown to the far left (NPBM1-5) next to CD14+ bone marrow mononuclear cells from myeloma patients (“MM” lanes). For the normal bone marrow control, CD14+ denotes the CD14+ mononuclear cell fraction (positive isolation) and CD14- denotes the CD14-depleted mononuclear fraction. Two different normal marrow donors gave identical results. C. Constitutive AKT activation at Ser473 in myeloma-associated monocytes/macrophages. The activation status of AKT was probed using antibodies against phosphorylated (phospho-Ser473 AKT) and total AKT. CD14+ peripheral blood monocytes from 5 different normal donors are shown to the far left (NPBM1-5) next to CD14+ bone marrow mononuclear cells from myeloma patients (“MM” lanes). For the normal bone marrow control, CD14+ denotes the CD14+ mononuclear cell fraction (positive isolation) and CD14- denotes the CD14-depleted mononuclear fraction. Two different normal marrow donors gave identical results. D. MAP3K8 expression levels are elevated in myeloma cells compared to non-lymphoid and non-haematopoietic tumour cells. MAP3K8 transcript expression data in multiple myeloma, leukaemia, lymphoma and solid tumour cell lines. Results are depicted in boxplots, which highlight the median and interquartile range of expression of MAP3K8 in each group. The whiskers of each boxplot represent the minimum and maximum expression of MAP3K8 for the respective group. The values for the log2-transformed median-centered expression of MAP3K8 transcript among myeloma cell lines was higher compared to solid tumours (p<0.0001, Kruskal-Wallis test for one-way analysis of variance, and p<0.05, post-hoc Dunn's multiple comparison test). E. MAP3K8 protein expression and ERK activation in primary myeloma tumour cells. Lysates from primary CD138+ myeloma tumour cells were immunoblotted using antisera against total MAP3K8, phospho-ERK, total ERK and GAPDH as a loading control. In myeloma cells, the p58 MAP3K8 isoform was preferentially expressed. F. MAP3K8 promotes ERK and JNK activation in response to TNF receptor stimulation in myeloma cells. MM1.S cells were serum-starved overnight in the presence or absence of MAP3K8 inhibitor (5 µM). A 5-min pulse of recombinant human TNFα (50 ng/ml) was given at the end of the incubation and cells were immediately lysed in protein buffer. MAP3K8 inhibition resulted in a reduction of phospho-ERK and phospho-JNK induction following stimulation with TNFα whereas p38MAPK activation status remained unaffected. G. Treatment of myeloma cells with a small-molecule MAP3K8 kinase inhibitor results in dose-dependent reduction in phosphorylation of the MAP3K8 direct substrate, MEK. U266 myeloma cells were incubated with escalating concentrations of MAP3K8 inhibitor for 24 h in the presence of continuous mitogenic stimulation (10% fetal calf serum). Whole cell lysates were subsequently immunoblotted and probed with antisera against phospho-MEK1/2 and total MEK1/2. DMSO, dimethyl sulfoxide.

Figure 2

A. MAP3K8 kinase inhibition results in dose-dependent growth inhibition of human myeloma cells. Human myeloma cell lines U266, MM1.S and RPMI8226 were plated at a starting concentration of 100,000 cells/ ml and treated with escalating concentrations of MAP3K8 kinase inhibitor as shown. Cultures were terminated at 96 h and viable cell counts performed. Each value represents the average of 6 counts obtained from 3 independent experiments, normalized to the untreated (DMSO-only) control. Error bars denote one standard deviation above and below the mean. Statistical significance is indicated with single asterisks at threshold p<0.05 and double asterisks at threshold p<0.01. B. MAP3K8 phosphorylation peaks in myeloma cells in mitosis. Cytospin preparations from U266 cells were immunostained using antisera against phospho-Thr290 MAP3K8 and total MAP3K8. Top left, actively growing U266 cells were stained with an antibody against phospho-Thr290 MAP3K8. Note that cytoplasmic staining correlates with mitotic nuclear morphology. Top right, U266 cells were pretreated with nocodazole overnight and immunostained with an anti-phospho-Thr290 antibody. Nocodazole inhibits spindle formation and results in G2/M arrest. Treatment with nocodazole results in cytoplasmic staining of numerous G2/M arrested cells. Middle left, actively growing U266 cells stained with an antibody against total MAP3K8. Almost all cells exhibit cytoplasmic staining, regardless of cell cycle status. Middle right, secondary antibody-only control. Bottom panels, cells were stained with antisera against phospho-Thr290 MAP3K8 (two different antibodies giving identical results, see Supplementary file for details) (cytoplasmic brown staining, DAB chromogen) followed by an antibody against phospho-Histone H3 (Ser10) (nuclear blue/violet staining, BCIP/NBT chromogen). Low and high power views are shown. Cells that exhibit cytoplasmic phospho-MAP3K8 staining also stain positive for nuclear phospho-Histone H3. Specific cytoplasmic staining for phospho-MAP3K8 is distinct from variable diffuse nuclear brown staining (background). C. Treatment of myeloma cells with a MAP3K8 kinase inhibitor results in apoptosis that is not rescued by the contact with myeloma-derived mesenchymal stromal cells. U266 myeloma cells were grown in the presence or absence of a monolayer of mesenchymal stromal cells derived from myeloma bone marrow. At 96 h of culture in the presence or absence of MAP3K8 inhibitor, cells were harvested, fixed and stained with Annexin V-fluorescein isothiocyanate and propidium iodide (PI) and analysed on a flow cytometer. Gates include early (Annexin V^high, PI^low) and late (Annexin V^high, PI^high) apoptotic cells. D. Cell-cycle profile of myeloma cells treated with a MAP3K8 kinase inhibitor. U266 myeloma cells were harvested at 96 h of culture in the presence or absence of MAP3K8 kinase inhibitor. Cells were fixed, stained with propidium iodide (PI) and analysed for DNA content on a flow cytometer. Treatment with MAP3K8 inhibitor results in an increase in the percentage of apoptotic cells (denoted by bar) without a corresponding G2/M arrest. E. Schematic diagram summarizing the complex actions of MAP3K8 kinase in the myeloma niche. In the proposed model, MAP3K8 participates in mitogenic signal transduction through the MAPK pathway and regulates TNF receptor-mediated inflammatory signal transduction in myeloma tumour cells. Additionally, MAP3K8 regulates production of TNFα (and other myeloma-promoting inflammatory cytokines) by myeloma-associated monocytes/macrophages through ERK-mediated mechanisms. MAP3K8 also controls AKT-mediated attenuation of macrophage inflammatory activity in order to avoid overt tissue injury by activated macrophages. Direct interactions are denoted by solid lines. Indirect interactions are denoted by broken lines.