Circumvention of Mcl-1-Dependent Drug Resistance by Simultaneous Chk1 and MEK1/2 Inhibition in Human Multiple Myeloma Cells

The anti-apoptotic protein Mcl-1 plays a major role in multiple myeloma (MM) cell survival as well as bortezomib- and microenvironmental forms of drug resistance in this disease. Consequently, there is a critical need for strategies capable of targeting Mcl-1-dependent drug resistance in MM. The present results indicate that a regimen combining Chk1 with MEK1/2 inhibitors effectively kills cells displaying multiple forms of drug resistance stemming from Mcl-1 up-regulation in association with direct transcriptional Mcl-1 down-regulation and indirect disabling of Mcl-1 anti-apoptotic function through Bim up-regulation and increased Bim/Mcl-1 binding. These actions release Bak from Mcl-1, accompanied by Bak/Bax activation. Analogous events were observed in both drug-naïve and acquired bortezomib-resistant MM cells displaying increased Mcl-1 but diminished Bim expression, or cells ectopically expressing Mcl-1. Moreover, concomitant Chk1 and MEK1/2 inhibition blocked Mcl-1 up-regulation induced by IL-6/IGF-1 or co-culture with stromal cells, effectively overcoming microenvironment-related drug resistance. Finally, this regimen down-regulated Mcl-1 and robustly killed primary CD138+MM cells, but not normal hematopoietic cells. Together, these findings provide novel evidence that this targeted combination strategy could be effective in the setting of multiple forms of Mcl-1-related drug resistance in MM

Bortezomib-resistant MM cells exhibiting Mcl-1 up-regulation and Bim down-regulation does not display cross-resistance to PD184352/CEP3891.

<p>(<b>A</b>) Parental U266 cells and their bortezomib-resistant counterparts (PS-R) were exposed to 20 nM bortezomib (btzmb) for 24 h, after which the percentage of apoptotic cells were determined by Annexin V staining and flow cytometry. Western blot analysis was performed to monitor Mcl-1 protein (inset). (<b>B</b>) U266 and PS-R cells were treated with 500 nM CEP3891±7.5 µM PD184352 for 48 h, after which the extent of apoptosis (Annexin V⁺ cells) was determined by flow cytometry. PS-R cells were exposed to a range of concentrations of CEP3891±PD184352 at a fixed ratio (1∶15) for 48 h, after which median dose effect analysis was used to characterize the nature of the interactions using cell death (7AAD⁺) as an endpoint (inset). (<b>C</b>) and (<b>D</b>) Alternatively, following treatment with 500 nM CEP3891±7.5 µM PD184352, real-time qRT-PCR and Western blot analysis were performed to monitor Mcl-1 mRNA levels (16 h, <b>C</b>) and expression of the indicated proteins (40 h, <b>D</b>). (<b>E</b>) and (<b>F</b>) In parallel, following 42 h-exposure to 500 nM CEP3891±7.5 µM PD184352, PS-R cells were subjected to immunoprecipitation (IP) followed by Western blotting to assess interactions between Mcl-1/Bim (<b>E</b>) and Mcl-1/Bak (<b>F</b>, upper), or Bak conformational change (<b>F</b>, lower). WCL was loaded to monitor protein levels. For analyses of flow cytometry and real-time qRT-PCR, values represent the means and SD for three separate experiments.</p

The PD184352/CEP3891 regimen down-regulates Mcl-1 and induces cell death in primary CD138⁺ MM cells.

<p>(<b>A</b>) and (<b>B</b>) Primary CD138⁺ MM cells (<b>A</b>) and their normal CD138⁻ counterparts (<b>B</b>) were isolated from bone marrow samples obtained from nine patients with MM, and exposed to 500 nM CEP3891±5 µM PD184352 for 24 h. After treatment, cell death was examined by trypan blue exclusion. (<b>C</b>) Alternatively, Western blot analysis was performed to monitor expression of Mcl-1 as well as cleavage of PARP and caspase 3 in the CD138⁺ and/or CD138⁻ populations. Each lane was loaded with 10 µg of protein. (<b>D</b>) A mechanistic model of circumvention of Mcl-1-dependent drug resistance by the Chk1/MEK inhibitor regimen. Mcl-1 plays an important role in both the survival of MM cell and sensitivity to various anti-MM agents, including bortezomib, as well as contributing to microenvironmental forms of drug resistance. A regimen combining a MEK1/2 inhibitor (MEKi) and a Chk1 inhibitor (Chk1i) acts at multiple levels in MM cells displaying Mcl-1-dependent bortezomib resistance, including a) down-regulation of Mcl-1 through a transcriptional mechanism; b) up-regulation of Bim and increased Bim/Mcl-1 binding, accompanied by release and activation of Bak and Bax; c) induction of MOMP (mitochondrial outer membrane permeabilization) and apoptosis; and d) possibly alternative Mcl-1-independent mechanism(s) of action (MOAs).</p

The PD184352/CEP3891 regimen increases Bim/Mcl-1 binding, releases Bak from Mcl-1, and triggers Bak/Bax activation.

<p>(<b>A</b>) and (<b>B</b>) U266 (<b>A</b>) and H929 (<b>B</b>) cells were exposed to 500 nM CEP3891±PD184352 (7.5 µM for U266; 2.5 µM for H929) for 42 h; (<b>C</b>) and (<b>D</b>) U266 cells over-expressing Mcl-1 and their EV controls were treated as described for U266 in panel 2A. After treatment, cells were lysed in 1% CHAPS buffer and immunoprecipitated (IP) using anti-Mcl-1 (<b>C</b>) or anti-Bak (<b>D</b>) antibodies, followed by Western blot (WB) analysis using anti-Bim, anti-Bak, or anti-Mcl-1 antibodies as indicated. WCL were loaded to monitor Bim levels. (<b>E</b>) and (<b>F</b>) Alternatively, following 24 h treatment, IP was performed to monitor conformational change of Bax and Bak using anti-Bax 6A7 or anti-Bak Ab-1 (for IP), and anti-Bax or anti-Bak (for WB) in parental U266 cells (<b>E</b>) and their counterparts ectopically expressing Mcl-1 (<b>F</b>). For all IP assays, IPs without cell lysate (-lysate) and/or with IgG (instead of primary antibodies) were carried out as controls; 200 µg protein per condition were employed for IP; IgG levels are shown to ensure equal loading of IP antibodies.</p

BMSCs fail to protect MM cells from PD184352/CEP3891 lethality.

<p><b>(A) and (B)</b> U226 cells stably expressing luciferase were co-cultured for 24 h with HS-5 cells (pre-cultured for 48 h), and then treated with either 50 µM dexamethasone (Dex) or 30 µM melphalan (Mel, <b>A</b>) or 400 nM CEP3891±7.5 µM PD184352 (<b>B</b>) for an additional 48 h. Bioluminescence intensity, which is proportional to the number of living cells, was monitored to assess cell viability. Values represent the means and SD for three separate experiments performed in triplicate. UT = untreated; RLU = relative light unit. (<b>C</b>) GFP-expressing U266 cells were co-cultured for 48 h with HS-5 cells (pre-cultured for 48 h) on the 4-well chamber slides, after which cells were treated with 400 nM CEP3891±7.5 µM PD184352 for an additional 40 h. Cells were then stained with 7AAD and images captured by an inverted fluorescence microscope (Olympus 1X71, 20× objective) with the filters suitable for 7AAD (red) or GFP (green). In parallel, bright field (BF) images were also captured for the same areas. (<b>D</b>) After treatment as described in panel 5B, GFP-expressing U266 cells were washed free of drugs and then plated with HS-5 cells on soft agar. After incubation for 21 days, the colony-forming ability of GFP⁺ U266 cells was assessed under fluorescence microscopy (Olympus 1X71, 4× objective); colonies were defined as clusters of >50 GFP⁺ cells. Bright field images were captured for comparison. The microscopic images are representative of three separate experiments. (<b>E</b>) H929 cells were treated with 300 nM CEP3891±2.5 µM PD184352 for 48 h under the conditions as follows: a) 10% FBS medium as control (lanes 1–4); b) HS-5-derived conditional medium (CM, lanes 5–8); and c) co-culture with HS-5 (lanes 9–12). In parallel, HS-5 cells alone (lanes 13–16) were treated for comparison. After drug treatment, Western blot analysis was conducted to monitor the expression of Mcl-1 and Bim, as well as caspase 3 cleavage.</p