A 13 mer LNA-i-miR-221 Inhibitor Restores Drug Sensitivity in Melphalan-Refractory Multiple Myeloma Cells

Abstract

Purpose: The onset of drug resistance is a major cause of treatment failure in multiple myeloma. Although increasing evidence is defining the role of miRNAs in mediating drug resistance, their potential activity as drug-sensitizing agents has not yet been investigated in multiple myeloma.

Experimental design: Here we studied the potential utility of miR-221/222 inhibition in sensitizing refractory multiple myeloma cells to melphalan.

Results: miR-221/222 expression inversely correlated with melphalan sensitivity of multiple myeloma cells. Inhibition of miR-221/222 overcame melphalan resistance and triggered apoptosis of multiple myeloma cells in vitro, in the presence or absence of human bone marrow (BM) stromal cells. Decreased multiple myeloma cell growth induced by inhibition of miR-221/222 plus melphalan was associated with a marked upregulation of pro-apoptotic BBC3/PUMA protein, a miR-221/222 target, as well as with modulation of drug influx-efflux transporters SLC7A5/LAT1 and the ABC transporter ABCC1/MRP1. Finally, in vivo treatment of SCID/NOD mice bearing human melphalan-refractory multiple myeloma xenografts with systemic locked nucleic acid (LNA) inhibitors of miR-221 (LNA-i-miR-221) plus melphalan overcame drug resistance, evidenced by growth inhibition with significant antitumor effects together with modulation of PUMA and ABCC1 in tumors retrieved from treated mice.

Conclusions: Taken together, our findings provide the proof of concept that LNA-i-miR-221 can reverse melphalan resistance in preclinical models of multiple myeloma, providing the framework for clinical trials to overcome drug resistance, and improve patient outcome in multiple myeloma.

Fig. 1. Correlation of melphalan sensitivity with induction of miR-221 and miR-222 expression in MM cell lines

A) U266/LR7, U266/S, RPMI-8226, NCI-H929, AMO1 and AMO1 Abzb cells were treated with increasing melphalan concentrations. Cell viability, relative to the untreated controls, was measured after 24h by CCK-8 assay. Values represent mean ± SD of 3 different experiments. B-C) qRT-PCR analysis of miR-221 (B) and miR-222 (C) expression was performed in all the six MM cell lines exposed to different melphalan concentrations (0–10–20 μM). Raw C_t data were normalized to RNU44 houskeeping gene and expressed as ΔΔC_t values, calculated using the comparative cross threshold (Ct) method. Values represent average ± SD of 3 independent experiments. P values (* p<0.05) were obtained using two-tailed t test.

Fig. 2. Anti-proliferative and apoptotic effects in MM cells of miR-221/222 inhibitors combined with melphalan

A) CCK-8 cell proliferation assay in U266/LR7, RPMI-8226 and AMO1 Abzb tranfected with 100 nM of miR-221/222 inhibitors or scrambled Control (NC) and then treated with increasing melphalan concentrations. Data shown are the average of three independent experiments, and p values were obtained using two-tailed test. (* p<0.05). B) Annexin V/7-AAD staining of U266/LR7, RPMI-8226 and AMO1 Abzb electroporated cells (miR-221/222 inhibitors or NC) 24h after treatment with different melphalan concentrations. The percentage of Annexin-V positive cells is plotted. Values represent the mean ± SD of 3 independent experiments. * p<0.05 C) Western blot analysis of total and cleaved Casp-8, Casp-9, Casp-3 and Cleaved PARP in U266/LR7 cells (top panel) and Casp-3 levels in RPMI-8226 cells (bottom panel). Cells lines were transfected with 100 nM of miR-221/222 inhibitors or NC and then exposed to melphalan (100μM for resistant U266/LR7 cells and 5μM for RPMI-8226 cells). Loading control was performed using GAPD or γ-tubulin. D) Fluorescence microscopy after Hoechst 33343 and PI staining of melphalan resistant U266/LR7 cells transfected with miR-221/222 inhibitors or NC, and then exposed to 100 μM of melphalan. DNA dye Hoechst stains the nucleus (blue color emission) of both viable and dead cells, allowing identification of nuclear morphology. Apoptotic nuclei appear fragmented and condensed, with greater signal intensity. Apoptotic and necrotic cells are identified as positive PI staining cells (red color emission) with loss of membrane integrity.

Fig. 3. Effects of miR-221/222 inhibitors plus melphalan on primary cells and on MM cell lines in the presence of BMSCs

A) CCK-8 cell proliferation assay in primary CD138+ MM patients cells tranfected with 100 nM of miR-221/222 inhibitors or scrambled Control (NC) and then treated with 100 uM of melphalan. Data shown are the average of two independent experiments, and p values were obtained using two-tailed test. (* p<0.05). B) CCK-8 cell proliferation assay in primary hPBMCs cells transfected with 100 nM of miR-221/222 inhibitors or scrambled Control (NC) and then treated with increased melphalan concentrations. Data shown are the average of three independent experiments, and p values were obtained using two-tailed test. (* p<0.05). C) 7-AAD staining of U266/LR7 cells transfected with 100 nM of miR-221/222 inhibitors or NC, and then cultured in the presence or absence of GFP+ hBMSCs. 24 hours after co-culture cells were treated with 100 μM of melphalan; 24h later, they were collected and stained for 7-AAD flow cytometry analysis. The percentage of 7-AAD positive cells is represented. Values represent the mean ± SD of 3 independent experiments. * p<0.05

Fig. 4. Inhibition of miR-221/222 enhances anti-MM activity of melphalan by modulation of PUMA/BBC3 expression

A) Dual-luciferase assay of U266/LR7 cells co-transfected either with NC or synthetic miR-221 or miR-222 mimics, together with firefly luciferase constructs containing the wild-type or mutant 3′ UTR of PUMA/BBC3 mRNA. The firefly luciferase activity was normalized to renilla luciferase activity. Data are represented as relative luciferase activity of either miR-221 or miR-222 mimics electroporated cells as compared to control. Values represent the mean ± SD of 3 independent experiments. * p<0.05 B) q-RT-PCR (bottom panel) and immunoblot (top panel) of PUMA/BBC3 in resistant U266/LR7 cells after transfection with NC or miR-221/222 inhibitors and treatment with 100 μM of melphalan. q-RT-PCR results are shown after normalization with GAPDH and ΔΔC_t calculation and represent an average ± SD of 3 independent experiments. Protein loading control for immunoblot was performed using GAPDH. C) q-RT-PCR (bottom panel) and immunoblot (top panel) of PUMA/BBC3 in sensitive U266/S cells after transfection with NC or miR-221/222 mimics and treatment with 60 μM of melphalan. q-RT-PCR results are shown after normalization with GAPDH and ΔΔC_t calculation and represent an average ± SD of 3 independent experiments. Protein loading control for immunoblot was performed using γ-tubulin. D) CCK-8 cell proliferation assay on resistant U266/LR7 cells (left panel) treated with either NC or miR-221/222 inhibitors co-transfected with siCNT or siBBC3/PUMA and subsequently exposed to 100 μM of melphalan. Effective knockdown was confirmed by immunoblot of PUMA/BBC3 protein and γ-tubulin normalization (right panel). Percentages of growth inhibition are plotted compared to control. Values represent the mean ± SD of 3 independent experiments. * p<0.05 E) CCK-8 cell proliferation assay on sensitive U266/S (left panel) transfected with siCNT or siBBC3/PUMA and treated with 60 μM of melphalan. Effective knockdown was confirmed by immunoblot of PUMA/BBC3 protein and GAPDH normalization (right panel). Cell viability is shown as percentage of control. Values represent the mean ± SD of 3 independent experiments. * p<0.05

Fig. 5. Effects of miR-221/222 inhibitors on the expression of drug influx-efflux transporters levels in resistant U266/LR7 cells

A) Western blot analysis of SLC7A5/LAT1 and ABCC1/MRP1 in U266/LR7 cells transfected with 100 nM of miR-221/222 inhibitors or NC and then exposed to 100μM of melphalan. ABBC1/MRP1 was immunoprecipitated from cell lysates (1mg) following incubation with 30 μl of protein A/G-conjugated agarose beads at 4°C overnight, and then detected by Western blot. Loading control was performed using γ-tubulin. B) Hoechst dye exclusion assay on U266/LR7 cells after treatment with either NC or miR-221/222 inhibitors or NC with Verapamil. The blue circles show the Side population (SP) with low levels of Hoechst fluorescence intensity. Dot plots represent one of three independent experiments. C) Bar column representation of percentage of SP affected after miR-221/222 inhibitors and Verapamil treatment as compared to NC. Values represent the mean ± SD of 3 independent experiments. * p<0.05

Fig. 6. LNA-i-miR-221 enhances anti-MM activity of melphalan overcoming drug-resistance

For the in vivo study, mice xenografted with U266/LR7 cells were randomized in 4 groups of treatment. A) Treatments were: i) i.p. LNA-i-miR-221 (25 mg/kg) at day 1–4–8–15–22; ii) scrambled control with the same schedule; iii) i.p. melphalan (0.75 mg/kg) on days 2–5–9–12–16–19; iiii) melphalan plus LNA-i-miR-221 with the above described schedules. B) mice were treated with: i) ip LNA-i-miR-221 (25 mg/kg) or ii) scramble control or iii) i.p melphalan (0.75 mg/kg) or iiii) melphalan plus LNA-i-miR-221 for 4 consecutive days every 10 days (–, –18). Arrows indicate the days of treatment. Tumors were measured with an electronic caliper every two days, and data are represented as averaged tumor volume ± SD of each group. P values were obtained using two-tailed t test and calculated by comparing LNA-i-miR-221 plus melphalan group versus each of the others. The pictures inserted show the in vivo detection of the tumor volume in a representative mouse of each group using IVIS LUMINA II Imaging System. * p < 0.05 ** p < 0.001 B) Western blot analysis of PUMA/BBC3 and ABBC1 levels in lysates from a representative retrieved xenograft from each treatment group. ABBC1/MRP1 was immunoprecipitated from cell lysates (1mg) following incubation with 30 μl of protein A/G-conjugated agarose beads at 4°C overnight. Immunoprecipitated ABCC1/MRP1 was detected by Western blot. Y-Tubulin was used as protein loading control. C) H&E (400x) and Ki-67 (400x) immunohistochemical analysis of retrieved xenografted tumors after different treatments. Representative images from each group are shown.