The IKK inhibitor Bay 11-7082 induces cell death independent from inhibition of activation of NFκB transcription factors.

Multiple myeloma (MM) displays an NFκB activity-related gene expression signature and about 20% of primary MM samples harbor genetic alterations conducive to intrinsic NFκB signaling activation. The relevance of blocking the classical versus the alternative NFκB signaling pathway and the molecular execution mechanisms involved, however, are still poorly understood. Here, we comparatively tested NFκB activity abrogation through TPCA-1 (an IKK2 inhibitor), BAY 11-7082 (an IKK inhibitor poorly selective for IKK1 and IKK2), and MLN4924 (an NEDD8 activating enzyme (NAE)-inhibitor), and analyzed their anti-MM activity. Whereas TPCA-1 interfered selectively with activation of the classical NFκB pathway, the other two compounds inhibited classical and alternative NFκB signaling without significant discrimination. Noteworthy, whereas TPCA-1 and MLN4924 elicited rather mild anti-MM effects with slight to moderate cell death induction after 1 day BAY 11-7082 was uniformly highly toxic to MM cell lines and primary MM cells. Treatment with BAY 11-7082 induced rapid cell swelling and its initial effects were blocked by necrostatin-1 or the ROS scavenger BHA, but a lasting protective effect was not achieved even with additional blockade of caspases. Because MLN4924 inhibits the alternative NFκB pathway downstream of IKK1 at the level of p100 processing, the quite discordant effects between MLN4924 and BAY 11-7082 must thus be due to blockade of IKK1-mediated NFκB-independent necrosis-inhibitory functions or represent an off-target effect of BAY 11-7082. In accordance with the latter, we further observed that concomitant knockdown of IKK1 and IKK2 did not have any major short-term adverse effect on the viability of MM cells

Multiple pilomatricomas with somatic CTNNB1 mutations in children with constitutive mismatch repair deficiency

Constitutional mismatch repair deficiency (CMMR-D) due to biallelic germline mutations in one of four mismatch repair genes causes a childhood cancer syndrome characterized by a broad tumor spectrum including hematological malignancies, and brain and Lynch syndrome-associated tumors. Herein, we report three children who had in addition to CMMR-D-associated malignancies multiple pilomatricomas. These are benign skin tumors of hair matrical differentiation frequently associated with somatic activating mutations in the ß-catenin gene CTNNB1. In two of the children, the diagnosis of CMMR-D was confirmed by the identification of biallelic germline PMS2 mutations. In the third individual, we only found a heterozygous germline PMS2 mutation. In all nine pilomatricomas with basophilic cells, we detected CTNNB1 mutations. Our findings indicate that CTNNB1 is a target for mutations when mismatch repair is impaired due to biallelic PMS2 mutations. An elevated number of activating CTNNB1 alterations in hair matrix cells may explain the development of multiple pilomatricomas in CMMR-D patients. Of note, two of the children presented with multiple pilomatricomas and other nonmalignant features of CMMR-D before they developed malignancies. To offer surveillance programs to CMMR-D patients, it may be justified to suspect CMMR-D syndrome in individuals fulfilling multiple nonmalignant features of CMMR-D (including multiple pilomatricomas) and offer molecular testing in combination with interdisciplinary counseling. © 2013 Wiley Periodicals, Inc.status: publishe

Functional titration of IKK inhibitors.

<p>(A,B) HT29 cells were pretreated for 1 h with the indicated concentrations of TPCA-1 and Bay 11-7082 and were subsequently challenged with TNF (50 ng/ml) for 3 and 10 min (A) or with Flag-TWEAK (200 ng/ml) for 8 hours (B). Total cell lysates were finally analyzed by Western blotting with respect to TNF-induced phosphorylation and degradation of IκBα (A) and TWEAK-induced p100 processing (B). (C) HT29 cells (40,000/chamber) were grown on glass slides and were pretreated with Bay 11-7082 (30 µM) or TPCA-1 (20 µM) for 30 min. Cells were then challenged with 100 ng/ml TNF for 1 h. After immunofluorescence staining for p65, the ratio of nuclear to cytoplasmic fluorescence intensity (FI) was determined. Data shown corresponds to 95–111 analyzed cells per experimental condition derived from a total of four independent experiments. (D) HT29 cells (20,000/well, 96 well-plate, triplicate values) were pretreated with the IKK inhibitors Bay 11-7082 (30 µM) or TPCA-1 (20 µM) for 30 min and were then stimulated with 100 ng/ml TNF for 6 h. The IL8 content of supernatants was subsequently determined by ELISA. To minimize the background signal related to constitutive IL8 production, medium was changed prior to inhibitor treatment. (E) The effects of TPCA-1 and Bay 11-7082 on TNF-induced phosphorylation and degradation of IκBα were analyzed in KMS-12-BM myeloma cells as described under “A”; n.s. = non specific. (F) Equivalent analysis on TNF-induced IL8 production as described in “D” using the RPMI8226 MM cell line. For statistical analysis of data shown in C, D and F one-way ANOVA with a Tukey post-test was performed. Asterisks indicate p-values≤0.01.</p

The mechanism of Bay 11-7082-induced MM cell death involves necrosis.

<p>(A) MM.1S and KMS-12-BM cells were treated with 30 µM Bay 11-7082 and analyzed by time-lapse video microscopy. Pictures shown represent typical stages of cells undergoing Bay 11-7082-induced cell death within 3 h. (B) Cells were pretreated for 30 min with BHA (50 µM), necrostatin-1 (90 µM) or remained untreated and were then challenged with 30 µM Bay 11-7082 for 2 h. Cells were finally photographed (B, arrows indicate swollen cells and the plasma membrane). (C) MM cells were either left untreated or pretreated for 1 h with BHA (50 µM), necrostatin-1 (90 µM), z-VAD-fmk (100 µM) or both necrostatin-1 and z-VAD-fmk. Cells were then challenged with 15 µM Bay 11-7082 for 1 h (KMS-12-BM) or 2 h (MM.1S) followed by annexin V-FITC/PI staining and FACS analysis. (D) MM.1S and KMS-12-BM cells were pretreated in triplicates for 30 min with the indicated combinations of BHA (50 µM), necrostatin-1 (90 µM) and z-VAD-fmk (100 µM), exposed for 2 h to 30 µM Bay 11-7082 and then analyzed for viability using the MTT assay. For statistical analysis a one-way ANOVA with a Tukey post-test was performed. Experimental settings that display significant protection against Bay 11-7082 induced cell death (p-values≤0.01) are indicated by asterisks.</p

Functional titration of MLN4924 and effects on MM cell viability.

<p>(A) HT29 and KMS-12-BM cells were pretreated for 1 h with the indicated concentrations of MLN4924 and challenged with TNF (50 ng/ml) for 3 and 10 min. Phosphorylation and degradation of IκBα were subsequently analyzed by Western blotting of total cell lysates. (B) HT29 cells (20,000/well, 96 well-plate, triplicate values) and RPMI8226 cells (50,000/well, 96 well-plate, triplicate values) were pretreated for 30 min with different concentrations of MLN4924. Cells were then stimulated with 100 ng/ml TNF for 6 h and the IL8 content of supernatants was determined by ELISA. For statistical analysis a one-way ANOVA with a Tukey post-test was performed. Groups showing significant inhibition (p-values≤0.01) of TNF-induced IL8 production are indicated by asterisks. (C) HT29 cells were pretreated for 1 h with the indicated concentrations of MLN4924 and then stimulated with Flag-TWEAK (200 ng/ml) for 18 h. The levels of p52, p100 and NIK were analyzed by Western blotting of total cell lysates. (D) MM cell lines were treated for 18 h with 10 µM MLN4924 and assayed for viability using the MTT assay. Significant (unpaired, two-tailed t-test, p-values≤0.01) induction of cell death is highlighted by asterisks. (E) Primary MM samples (n = 11) in co-culture with BMSCs were treated with 10 µM MLN4924 for 3 days and cell death was assessed by annexin V-FITC/PI staining and FACS analysis.</p

Knockdown of IKK1, IKK2 or both in MM cells.

<p>(A) Western blots showing depletion of IKK1 and IKK2 in L363 cells after single or combined transfection with stealth siRNAs for the respective targets. Scram denotes non-specific stealth siRNA control. Samples prepared at day 2 post-electroporation from cells co-transfected with an expression plasmid for CD4Δ and purified by CD4 microbead selection of strongly transfected cells. (B) Viability (AlamarBlue, left panel) and survival (annexin V, right panel) assays measured at day 4 post-electroporation for the sample preparations shown in “A”. All values calculated relative to the measurement obtained for the non-specific siRNA control at 2.5 µM concentration (i.e. the concentration present in single stealth siRNA transfections). (C) Overview of the viability and survival effects of IKK1 and IKK2 knockdown in MM cell lines JJN3, L363 and MM.1S. Mean values (calculated with respect to the respective non-specific siRNA controls) and standard deviations shown.</p

Effects of IKK inhibitors on MM cell viability.

<p>(A,B) MM cell lines were challenged with either TPCA-1 (20 µM) or Bay 11-7082 (30 µM) for 4 h (A) and 18 h, respectively (B), and analyzed for viability using the MTT assay. (C) Primary MM samples (n = 11) in co-culture with BMSCs were incubated with either TPCA-1 (20 µM) or Bay 11-7082 (30 µM) for 3 days and cell death was determined by annexin V-FITC/PI staining and flow cytometry (relative values with respect to the DMSO-treated controls shown; horizontal lines indicate the mean). Data were analyzed using a two-tailed, paired t-test. Asterisk indicates a p<i>-</i>value <0.01. (D) MM.1S and KMS-12-BM cells were challenged with 30 µM Bay 11-7082 for the indicated time. Triplicate values were taken and cellular viability was determined using the MTT assay.</p