In vitro and in vivo selective antitumor activity of a novel orally bioavailable proteasome inhibitor MLN9708 against multiple myeloma cells

Abstract

Purpose: The success of bortezomib therapy for treatment of multiple myeloma (MM) led to the development of structurally and pharmacologically distinct novel proteasome inhibitors. In the present study, we evaluated the efficacy of one such novel orally bioactive proteasome inhibitor MLN9708/MLN2238 in MM using well-established in vitro and in vivo models.

Experimental design: MM cell lines, primary patient cells, and the human MM xenograft animal model were used to study the antitumor activity of MN2238.

Results: Treatment of MM cells with MLN2238 predominantly inhibits chymotrypsin-like activity of the proteasome and induces accumulation of ubiquitinated proteins. MLN2238 inhibits growth and induces apoptosis in MM cells resistant to conventional and bortezomib therapies without affecting the viability of normal cells. In animal tumor model studies, MLN2238 is well tolerated and inhibits tumor growth with significantly reduced tumor recurrence. A head-to-head analysis of MLN2238 versus bortezomib showed a significantly longer survival time in mice treated with MLN2238 than mice receiving bortezomib. Immununostaining of MM tumors from MLN2238-treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Mechanistic studies showed that MLN2238-triggered apoptosis is associated with activation of caspase-3, caspase-8, and caspase-9; increase in p53, p21, NOXA, PUMA, and E2F; induction of endoplasmic reticulum (ER) stress response proteins Bip, phospho-eIF2-α, and CHOP; and inhibition of nuclear factor kappa B. Finally, combining MLN2238 with lenalidomide, histone deacetylase inhibitor suberoylanilide hydroxamic acid, or dexamethasone triggers synergistic anti-MM activity.

Conclusion: Our preclinical study supports clinical evaluation of MLN9708, alone or in combination, as a potential MM therapy.

Figure 1. Proteasome inhibitor MLN2238 is structurally distinct from bortezomib, and inhibits proteasome activity in vitro

(A–C) MM.1S cells were treated with MLN2238 at indicated concentrations for 3h and harvested; cell extracts were then analyzed for CT-L, C-L, and T-L proteasome activities. Results are represented as percent inhibition in proteasome activities in drug-treated vs. vehicle control. (D) MM.1S cells were treated with IC₅₀ concentrations of MLN2238 or bortezomib for indicated times and harvested; cell extracts were then analyzed for CT-L proteasome activity. (E, left panel) MM.1S cells were treated with MLN2238 (12 nM) at indicated times and harvested; protein lysates were subjected to immunoblotting using anti-ubiquitin and anti-actin Abs. Blots shown are representative of 3 independent experiments. (E, right panel) MM.1S cells were treated with MLN2238 for 24h and harvested; protein lysates were subjected to immunoblotting using anti-ubiquitin and anti-actin Abs. Blots shown are representative of 3 independent experiments. (F) Recombinant human HtrA2 enzyme was incubated with its substrate β-casein in assay buffer (R&D Systems), followed by SDS-PAGE, silver staining, and quantification of cleaved β-casein. The bar graph represents percent inhibition of HtrA2-induced β-casein cleavage in the presence of bortezomib (3 nM) or MLN2238 (12 nM). Data presented are means plus or minus SD (n=2; P < 0.05).

Figure 2. Anti-MM activity of MLN2238

(A) MM cell lines were treated with or without MLN2238 at the indicated concentrations for 48h, followed by assessment for cell viability using MTT assays. Data presented are means plus or minus SD (n =3; P < 0.05 for all cell lines). (B) MM.1S, H929, OPM1, and OPM2 cell lines were treated with MLN2238 (IC₅₀ concentrations) and analyzed for apoptosis using Annexin V/PI staining assay. (C) Purified patient MM cells (CD138-positive) were treated with indicated concentrations MLN2238 for 24h and 48h, and cell viability was measured using Celltiter Glo assay. Data presented are means plus or minus SD of triplicate samples (P < 0.001 for all patient samples). (D) Bortezomib-sensitive (ANBL-6.WT) and -resistant (ANBL-6.BR) MM cell lines were treated with increasing concentrations of bortezomib and MLN2238 for 48h, followed by assessment for cell viability using MTT assays. The IC₅₀ of MLN2238 and bortezomib for ANBL-6.WT or ANBL-6.BR was derived. The bar graph shows the IC₅₀ ratio (ANBL-6.BR/ANBL-6.WT) of ML2238 and bortezomib. Data presented are means plus or minus SD (n =3). (E) PBMCs from healthy donors were treated with indicated concentrations of MLN2238 for 48h, and then analyzed for viability using Celltiter Glo assay. Data presented are means plus or minus SD of quadruplicate samples (P < 0.001 for all PBMCs samples).

Figure 3. MLN2238 inhibits growth of xenografted human MM cells in CB-17 SCID mice

(A) Average and standard deviation of tumor volume (mm³) is shown from mice (n = 7/group) versus time (days) when tumor was measured. MM.1S cells (5 × 10⁶ cells/mouse) were implanted in the rear flank of female mice (6 weeks of age at the time of tumor challenge). On Day 28–30, mice were randomized to treatment groups and treated intravenously with vehicle, MLN2238 (11 mg/kg), or with bortezomib (1 mg/kg) on a twice weekly schedule for 3 weeks. Data are presented as mean tumor volume ± SD. A significant delay in tumor growth in MLN2238-treated mice was noted compared to vehicle-treated control mice. Bars indicate means plus or minus SD. (B) Kaplan-Meier survival plot shows significant increase (P < 0.05) in survival of mice receiving MLN2238 (11 mg/kg) or bortezomib (1 mg/kg) compare to vehicle treated control. (C) Mice were treated with vehicle, MLN2238, or bortezomib (as in panel A); blood samples were obtained and subjected to analysis for bilirubin, creatinine, and hemoglobin levels using Quantichrom™ Creatinine, Bilirubin, and Hemoglobin Assay kit (BioAssay Systems, Hayward, CA). (D) Micrographs (upper three panels) show apoptotic cells in tumors sectioned from untreated- or MLN2238-treated mice as identified caspase-3 cleavage (Red cells), TUNEL (TUNEL-positive cells: Green cells), as well as Ki67 staining. Dotted red/green line indicates a line between tumor and host tissue. Micrographs (lower two panels) show expression of angiogenesis markers in tumors sectioned from untreated- or MLN2238-treated mice, identified by VEGFR2 (Green) and Pecam (Red color) staining. Bar scale: 100 µm (caspase-3 and TUNEL); 50 µm (Ki67); 10 µm (VEGFR2 and Pecam). (E) Tumor-bearing mice were treated orally with vehicle, or MLN2238 (8 mg/kg) on a twice weekly schedule for 3 weeks. Data are presented as mean tumor volume ± SD. Bars indicate means plus or minus SD. (F) Kaplan-Meier survival plot shows increase in survival of mice receiving oral doses of MLN2238 (8 mg/kg) compare to vehicle treated control.

Figure 4. Mechanisms mediating anti-MM activity of MLN2238

(A and B) H929 and MM.1S and cells were treated with MLN2238 at the indicated doses for 24h and harvested; whole cell lysates were subjected to immunoblot analysis with anti-PARP, anti-caspase-3, anti-caspase-8, anti-caspase-9, or anti-actin Abs. FL indicates full length; CF denotes cleaved fragment. (C) MM.1S cells were pretreated with biochemical inhibitors of caspase-8 (IETD-FMK), caspase-9 (LEHD-FMK), or pan-caspase (Z-VAD-FMK) for 1h. MLN2238 (12 nM) was then added in cultures for additional 24h, followed by analysis of cell viability by MTT assay. Data presented are means plus or minus SD (n = 3; P = 0.005). Error bars represent standard deviation (SD). (D and E) MM.1S cells were treated with MLN2238 (12nM) for 24h and harvested; protein lysates were subjected to immunoblotting using indicated Abs. (F) MM.1R cells were treated with MLN2238 (12nM) for 24h and harvested; protein lysates were subjected to immunoblotting using indicated Abs. (G) MM.1S cells were treated with MLN2238 (12 nM) for indicated times and harvested; protein lysates were subjected to immunoblotting using indicated Abs. Blots shown in the figure are representative of 3 independent experiments.

Figure 5. MLN2238 blocks BMSCs-induced MM cell proliferation, inhibits in vitro capillary tubule formation, and target NF-κB

(A) MM.1S cells were treated with indicated concentrations of MLN2238 in the presence or absence of BMSCs for 48h, followed by measurement of proliferation using tritiated thymidine incorporation assay. Data presented are means plus or minus SD (n =3; P < 0.005). (B) Human Umbilical Vein Endothelial Cells (HUVEC) were cultured in the presence or absence of MLN2238 for 8h (cell viability > 95%); cells were then washed with plain media, and placed on the matrigel for 4h to allow for tube formation, followed by photography using inverted microscope (magnification: 4X/0.10 NA oil, media: EBM-2). The in vitro angiogenesis is reflected by capillary tube branch formation. Images are representative of 3 experiments with similar results. (C and D) MM.1S cells were treated with MLN2238 (12 nM) at indicated times, harvested; and subjected to NF-κB activity analysis using ELISA. Data presented are the means plus or minus SD (n = 3; P < 0.005). (E and F) MM.1S cells were treated with MLN2238 (12 nM) at indicated times, followed by addition of TNF-α during the last 20 mins before cells were harvested. Cells were then subjected to p65 and p52 NF-κB activity analysis using ELISA. Data presented are the means plus or minus SD (n = 3; P < 0.004). Error bars represent standard deviation.

Figure 6. Combination of low doses of MLN2238 and lenalidomide, SAHA, or Dex trigger synergistic anti-MM activity

(A) MM.1S cells were treated for 48h with indicated concentrations of MLN2238, lenalidomide, or MLN2238 plus lenalidomide; and then assessed for viability using MTT assays. Isobologram analysis shows the synergistic cytotoxic effect of MLN2238 and lenalidomide. The graph (left panel) is derived from the values given in the Table (right panel). The numbers 1–9 in graph represent combinations shown in the Table. Fa_Com = Fraction of cells showing decrease in viability with MLN2238 plus lenalidomide treatment. Combination index (CI) of < 1 indicates synergy. All experiments were performed in triplicate and mean value is shown. (B) MM.1S cells were treated for 48h with indicated concentrations of MLN2238, SAHA, or MLN2238 plus SAHA; and then assessed for viability using MTT assays. Isobologram analysis shows the synergistic cytotoxic effect of MLN2238 and SAHA. The graph (left panel) is derived from the values given in the Table (right panel). The numbers 1–9 in graph represent combinations shown in the Table. Combination index (CI) of < 1 indicates synergy. (C) MM.1S cells were treated for 48h with indicated concentrations of MLN2238, Dex, or MLN2238 plus Dex; and then assessed for viability using MTT assays. Isobologram analysis shows the synergistic cytotoxic effect of MLN2238 and Dex. The graph (left panel) is derived from the values given in the Table (right panel).