Proteasome inhibition in myelodysplastic syndromes and acute myelogenous leukemia cell lines

Abstract

In this work, effects of bortezomib on apoptosis, clonal progenitor growth, cytokine production, and NF-κB expression in patients with MDS with cytopenias requiring transfusion support are examined. Bortezomib increased apoptosis in marrow mononuclear cells but had no effects on CFU-GM, BFU-E, or CFU-L content. No consistent effects on NF-κB activation in vivo were noted. To further define the role of bortezomib in AML and MDS, we examined it in combination with several targeted agents and chemotherapeutic agents in vitro. Combinations with arsenic trioxide, sorafenib, and cytarabine demonstrated synergistic in vitro effects in AML cell lines.

Trial registration: ClinicalTrials.gov NCT00262873.

Figure 1

Effect of in vivo bortezomib on apoptosis. Marrow was sampled from patients enrolled on the clinical trial before bortezomib exposure and at day 14 after four doses of bortezomib. Shown is the percentage of light density cells in apoptosis as assessed by Annexin V staining. Data are shown as Mean ± SEM for the five pre and postbortezomib samples. N = 5; p = .05 by paired t-test.

Figure 2

Effect of in vivo bortezomib on progenitor cells. In vivo exposure to bortezomib did not significantly affect the outgrowth of clonogenic progenitors (CFU-GM (A), BFU-E (B), or CFU-L(C)) between screening marrow (day 0) and 14 days after the start of bortezomib. N = 5. Data shown are individual values and means ± SD.

Figure 3

Effects of bortezomib on IL-6 and VEGF levels. Fourteen days after the start of bortezomib, the serum levels of IL-6 had not changed significantly, whereas the levels of VEGF were significantly decreased (p < .05). n = 5. Bars shown are Mean ± SD.

Figure 4

Expression of active NF-κB in MDS patients. Shown is the degree of active NF-κB expression at baseline in the blood of six MDS patients. p65 NF-κB activity was assayed using the Trans-AM assay kit and quantified by ELISA. The Y-axis represents OD units.

Figure 5

Effect on expression of active NF-κB after 14 days of bortezomib therapy. Shown is the pre and postlevels of expression of active NF-κB after 14 days of bortezomib. P65 NF-κB activity was assayed using the Trans-AM assay kit and quantified by ELISA. The Y-axis represents OD units. Data from patients 1 through 3 is from paired marrow samples, and data from patient 4 is from blood due to inaspirable marrow.

Figure 6

Time course of expression of active NF-κB in blood mononuclear cells after exposure to bortezomib. In this patient, an initial increase in activation was followed by a decline after additional cycles of bortezomib were administered.

Figure 7

Effect of concurrent bortezomib and arsenic trioxide on proliferation of HL60 cells as measured by MTT. Bortezomib (1 nM) and arsenic (0.5 μM) were added to HL60 cells, and proliferation was determined by MTT assay at 48 hr. Data shown is Mean ± SEM with n = 3.

Figure 8

Effect of bortezomib and arsenic trioxide on phospho-ERK and phospho-AKT expression in the HL60 cell line. Proteins were extracted, electropheresed as described, and probed with the appropriate antibody. This experiment was completed twice in the HL60 cell line with comparable results and normalization to an actin control (not shown given equidensity ERK bands).

Figure 9

Effect of the farnesyltransferase inhibitor, L-744832 on levels of active NF-κB expression as measured by ELISA for the p65 component in the HL60 cell line.

Figure 10

The combination of sorafenib and bortezomib inhibited proliferation in AML cells lines beyond that seen with either agent alone (A). Apoptosis was also increased with the combination as compared with the control (B). Shown is the Mean ± SD of cell proliferation as measured in the MTT assay (OD) or percentage of cells in apoptosis as measured by Annexin V at 48 hr in the MV411 cell line. (n = 3 independent experiments).

Figure 11

Effect of concurrent cytarabine and bortezomib exposure on primary AML blasts. Concurrent exposure decreased proliferation as compared with either agent alone. Shown is a representative experiment of 3. Similar effects were noted with leukemia cell lines. B = bortezomib.

Figure 12

Effect of cytarabine (ARA-C) and bortezomib on expression of cleaved PARP and activated caspase-3 in a primary AML sample. Beta-actin served as a control to assure equal protein loading.

Figure 13

Effect of 5-azacytidine(Vidaza^™) (V) and bortezomib (B) on viability in the KG1a leukemia cell line. Cells were cultured in plastic flasks for 24–72 hr. No significant effects of the combination were noted. N = 3.

Figure 14

Effect of 5-azacytidine (V) and bortezomib (V) on viability in the MV-411 cell line. The combination did not result in increased apoptosis (viability measured as Annexin V expression) as compared with the control conditions. N = 3.