Targeting NF-kappaB pathway with an IKK2 inhibitor induces inhibition of multiple myeloma cell growth

Abstract

The pathophysiologic basis for multiple myeloma (MM) has been attributed to the dysregulation of various paracrine or autocrine growth factor loops and to perturbations in several signal transduction pathways including IkappaB kinase/nuclear factor-kappaB (IKK/NF-kappaB). The present study aimed at investigating the effect of a pharmaceutical IKK2 inhibitor, the anilinopyrimidine derivative AS602868, on the in vitro growth of 14 human MM cell lines (HMCL) and primary cells from 13 patients. AS602868 induced a clear dose-dependent inhibition of MM cell growth on both HMCL and primary MM cells, which was the result of a simultaneous induction of apoptosis and inhibition of the cell cycle progression. Combination of AS602868 with suboptimal doses of melphalan or Velcade showed an additive effect in growth inhibition of HMCL. AS602868 also induced apoptosis of primary myeloma cells. Importantly, AS602868 did not alter the survival of other bone marrow mononuclear cells (CD138(-)) co-cultured with primary MM (CD138(+)) cells, except for CD34(+) haematopoietic stem cells. The results demonstrate the important role of NF-kappaB in maintaining the survival of MM cells and suggest that a pharmacological inhibition of the NF-kappaB pathway by the IKK2 inhibitor AS602868 can efficiently kill HMCL and primary myeloma cells and therefore might represent an innovative approach for treating MM patients.

Fig 1. Inhibition of NF-kB activation in HMCL by AS602868 IKK2 inhibitor

(A) Western blot analysis of IκBα activation. XG-6 and XG-14 cell lines were IL-6-starved in culture medium with 2 % FCS for 3 hours in order to reduce the constitutive NF-κB activation. Then, cells were pre-treated for 30 min_with or without AS602868 (AS68) (10 μM) before stimulation with TNF-α (10 ng/ml) for 15 min. Cells were harvested for Western blot analysis. Blots were probed with anti-phospho-IκBα and anti-IκBα antibodies. (B) Gene expression analysis by quantitative RT-PCR (Q-RT-PCR). XG-6 and XG-14 HMCL were IL-6-starved to reduce constitutive NF-κB activation and pre-treated with or without AS602868 as in (A) before stimulation with TNF-α (10 ng/ml) for one hour. The relative gene RNA level was determined as described in Materials and Methods. The “1” value was assigned for each cell line to the control before pre-treatment with AS602868.

Fig 2. Inhibition of the HMCL proliferation by AS602868 IKK2 inhibitor

HMCL were cultured for 4 days in the presence of graded concentrations of AS602868 IKK2 inhibitor. Mean values of [³H]-thymidine incorporation were determined in triplicate culture wells and results are expressed as percentages of the mean proliferation without inhibitor (control culture medium). Results are those of one representative experiment out of a minimum of two for each cell line. The mean thymidine incorporation without AS602868 inhibitor was 32823 cpm for XG-2, 100434 for XG-7, 102505 for XG-12, 35294 for XG-14, 56981 for RPMI8226, 38677 for XG-1, 127328 for XG-3, 22309 for XG-11, 23535 for XG-20 and 61951 for L363.

Fig 3. AS602868 IKK2 inhibitor inhibits the cell survival of primary myeloma cells in culture bone marrow

Bone marrow mononuclear cells from 6 newly-diagnosed myeloma (A) and 7 relapsing patients (B) were isolated as described in Material and methods and cultured without (control culture medium) or with graded concentrations of AS602868 IKK2 inhibitor. At day 5 of culture, the number of viable cells in each group of culture was assessed by trypan blue exclusion, the cells were stained with anti-CD138 and anti-CD34 antibodies and the percentage of CD138 and CD34 cells determined by flow cytometry. The number of CD138+, CD138− and CD34+ viable cells was then calculated. Results are expressed as the relative number of viable cells from each cell subpopulation obtained with AS602868 compared to that obtained in the corresponding subpopulation in the presence of control culture medium. Statistical analyses were carried out using a Student’s t test for pairs. * Significantly lower values than control (P < .05); ** significantly lower values than control (P < .01).

Fig 4. Sensitivity of HMCL to AS602868 IKK2 inhibitor without or with IC10 or IC50 melphalan or Velcade

HMCL were cultured as indicated in Fig 2 in the presence of graded concentrations of AS602868, without (control culture medium) or with the respective IC10 and IC50 concentrations of melphalan or Velcade for each cell line. IC50 and IC90 for AS602868 without or with melphalan or Velcade were then deduced.