Blockade of the MEK/ERK signalling cascade by AS703026, a novel selective MEK1/2 inhibitor, induces pleiotropic anti-myeloma activity in vitro and in vivo

Abstract

This study investigated the cytotoxicity and mechanism of action of AS703026, a novel, selective, orally bioavailable MEK1/2 inhibitor, in human multiple myeloma (MM). AS703026 inhibited growth and survival of MM cells and cytokine-induced osteoclast differentiation more potently (9- to 10-fold) than AZD6244. Inhibition of proliferation induced by AS703026 was mediated by G0-G1 cell cycle arrest and was accompanied by reduction of MAF oncogene expression. AS703026 further induced apoptosis via caspase 3 and Poly ADP ribose polymerase (PARP) cleavage in MM cells, both in the presence or absence of bone marrow stromal cells (BMSCs). Importantly, AS703026 sensitized MM cells to a broad spectrum of conventional (dexamethasone, melphalan), novel or emerging (lenalidomide, perifosine, bortezomib, rapamycin) anti-MM therapies. Significant tumour growth reduction in AS703026- vs. vehicle-treated mice bearing H929 MM xenograft tumours correlated with downregulated pERK1/2, induced PARP cleavage, and decreased microvessels in vivo. Moreover, AS703026 (<200 nmol/l) was cytotoxic against the majority of tumour cells tested from patients with relapsed and refractory MM (84%), regardless of mutational status of RAS and BRAF genes. Importantly, BMSC-induced viability of MM patient cells was similarly blocked within the same dose range. Our results therefore support clinical evaluation of AS703026, alone or in combination with other anti-MM agents, to improve patient outcome.

Fig. 1. AS703026 inhibited growth and survival of MM cell lines as well as osteoclast formation

A, The chemical structure of AS703026. B, INA-6 MM cells were treated with AS703026 or AZD6244 for 2 days, followed by [³H]thymidine uptake assay. C, MM cells, sensitive or resistant to standard of care agents, were incubated with DMSO or a dose range of AS703026 (2-2000 nM) for 2 days, and subjected to DNA synthesis assay. D, PBMCs isolated from normal donors (n=3) were incubated with M-CSF and RANKL, in the presence or absence of AS703026 or AZD6244 for 14 days. The TRAP assay was performed to measure the formation of multinuclear osteoclast cells (OC).

Fig. 2. AS703026 specifically blocked ERK1/2 activation in MM cells, cultured alone or with BMSCs

A, U266 cells were incubated with DMSO or AS703026 (5-2000 nM) for 1h (left) or 200 nM for 0-72h (right) and then subjected to immunoblotting. B, MM cell lines cultured alone or with BMSCs were treated with (+) or without (-) AS703026 (5 μM for MM1S; 0.5 μM for H929) overnight, followed by immunoblotting of cell lysates with indicated Abs. Anti-α-tubulin was used as a loading control.

Fig. 3. AS703026 induced apoptosis and modulated the cell cycle profile

A, H929 MM cells were treated with AS703026 (0.5 μM) for 0-72h; cell lysates were then subjected to immunoblotting using specific Abs. B, H929 MM cells were treated with AS703026 (0.1 μM) and stained with PI; cell cycle profiles were evaluated using flow cytometry.

Fig. 4. AS703026 targeted MM cells in the BM microenvironment

MM cells, cultured alone or with BMSCs, were treated with DMSO or a dose range of AS703026 (0.002-20 μM) and conditioned media were collected for measurement of VEGF (A) and IL-6 (B) concentrations by ELISA. p<0.05 for AS703026 >20 nM vs control. C, Conditioned media from MM patient cells cultured with or without BMSCs were similarly examined for IL-6. p<0.02 (left panel). IL-6 from BMSCs alone was reduced when higher concentrations (>2000 nM) of AS703026 were used (right panel).

Fig. 5. AS703026 combined with anti-MM drugs augmented MM cell death

A, MM1S cells, which are relatively insensitive to AS703026, were treated with DMSO or a dose range of AS703026 (0.02-20 μM) and rapamycin (0.1-100 nM) for 2 days, followed by DNA synthesis assay. B, U266 cells were treated with AS703026 (20 nM) or dexamethasone (dex; 0.1, 1 μM) alone or in combination for 3 days, followed by annexin V/PI staining and flow cytometric analysis. C, Dex-resistant CD138+ patient MM cells were cocultured with BMSCs for 5 days with AS703026 (200 nM), dex (1, 10 μM), or the combination, and then subjected to apoptosis assay. Dex- and AS703026-resistant MM1R cells were treated with AS703026 (200 nM) with or without melphalan (mel 0.2, 2 μM; D) or lenalidomide (len; E), alone or together for 5 days. Control medium, thin line; treatment, bold line

Fig. 5. AS703026 combined with anti-MM drugs augmented MM cell death

A, MM1S cells, which are relatively insensitive to AS703026, were treated with DMSO or a dose range of AS703026 (0.02-20 μM) and rapamycin (0.1-100 nM) for 2 days, followed by DNA synthesis assay. B, U266 cells were treated with AS703026 (20 nM) or dexamethasone (dex; 0.1, 1 μM) alone or in combination for 3 days, followed by annexin V/PI staining and flow cytometric analysis. C, Dex-resistant CD138+ patient MM cells were cocultured with BMSCs for 5 days with AS703026 (200 nM), dex (1, 10 μM), or the combination, and then subjected to apoptosis assay. Dex- and AS703026-resistant MM1R cells were treated with AS703026 (200 nM) with or without melphalan (mel 0.2, 2 μM; D) or lenalidomide (len; E), alone or together for 5 days. Control medium, thin line; treatment, bold line

Fig. 6. AS703026 inhibited tumor growth in a human plasmacytoma model of H929 MM cells

A, Mice bearing H929 MM tumors were treated with control vehicle (0.5%cmc/0.25%Tween20; n = 6) or AS703026 (n = 6 for 15 mg/kg, n = 4 for 30 mg/kg) BID orally (5 days per week for 2 weeks). Animals were euthanized when tumors reached 2 cm³ in size. Treatment with AS703026 inhibited tumor growth compared to vehicle control (RM-ANOVA & Fisher's LSD, p < 0.05) B, Four hours after the last dose, tumors were excised and subject to immunoblotting for pERK1/2 and cleaved PARP. Immunoblotting for ERK1/2 confirmed equal protein loading. C, IHC studies of tumors for pERK, cleaved caspase3, and TUNEL (200× magnification) and D, CD34 (100× magnification) Images were taken using a Leica DFC300FX with a 10/0.22 NA objective and a Leica IM50 Image Manager.

Fig. 7. AS703026 is cytotoxic against CD138-purified MM cells from patients with relapsed and refractory MM

CD138-purified patient tumor cells were treated with AS703026 for 5 days and then subjected to MTT (A) or [³H]thymidine uptake (B) assays. Data in A and B were derived from the same patient cells (MM1-8). Two patient MM cells (MM11 & MM14) were also cocultured with BMSCs in the presence or absence of drug for 5 days (C).