ALCAM regulates multiple myeloma chemoresistant side population

Abstract

Drug-resistance is a major problem preventing a cure in patients with multiple myeloma (MM). Previously, we demonstrated that activated-leukocyte-cell-adhesion-molecule (ALCAM) is a prognostic factor in MM and inhibits EGF/EGFR-initiated MM clonogenicity. In this study, we further showed that the ALCAM-EGF/EGFR axis regulated the MM side population (SP)-mediated drug-resistance. ALCAM-knockdown MM cells displayed an enhanced ratio of SP cells in the presence of bone marrow stromal cells (BMSCs) or with the supplement of recombinant EGF. SP MM cells were resistant to chemotherapeutics melphalan or bortezomib. Drug treatment stimulated SP-genesis. Mechanistically, EGFR, primed with EGF, activated the hedgehog pathway and promoted the SP ratio; meanwhile, ALCAM inhibited EGFR downstream pro-MM cell signaling. Further, SP MM cells exhibited an increased number of mitochondria compared to the main population. Interference of the mitochondria function strongly inhibited SP-genesis. Animal studies showed that combination therapy with both an anti-MM agent and EGFR inhibitor gefitinib achieved prolonged MM-bearing mice survival. Hence, our work identifies ALCAM as a novel negative regulator of MM drug-resistance, and EGFR inhibitors may be used to improve MM therapeutic efficacy.

Fig. 1. Bone marrow stroma-derived soluble factors promote ALCAM-knockdown myeloma cell side population.

A Hoechst staining of CTR-KD or AL-KD MM cells (RPMI8226 or MM.1S) cultured in regular medium (CTR-M) or BMSC-conditioned medium (BMSC-M) for 48 h. The numbers indicate the percentage of SP cells. B Hoechst staining of AL-KD1 and AL-KD2 RPMI8226 cells cultured in regular medium (CTR-M) or BMSC-conditioned medium (BMSC-M) for 48 h. C Schematic graph: After cell sorting, SP cells were labeled with red fluorescent dye, and MP cells were labeled with CFSE. The labeled SP and MP cells were re-mixed with the ratio (SP:MP = 2:98). D SP cell proliferation was examined by flow cytometry for red fluorescent intensity dilution. E Fluorescence confocal microscopy of MM cells (green) and BMSCs (red) under 3D printing. F Hoechst staining of RPMI8226 cells, followed by cell sorting to isolate SP- and MP-only MM cells. The cells were used for the set of colony formation assay (left panel). Statistical results from the colony formation assays (right panel). Data are the mean of three independent experiments in three replicates. *p < 0.05.

Fig. 2. ALCAM-EGF/EGFR axis regulates myeloma side population.

A Hoechst staining of RPMI8226 cells in the presence of an EGF neutralizing antibody (10 μg/ml) for 24 h and the result quantification (right panel). B Hoechst staining of CTR-KD or AL-KD RPMI8226 cells in the presence of recombinant EGF protein (10 ng/ml) for 24 h and the result quantification (right panel). C Hoechst staining of AL-KD1 and AL-KD2 RPMI8226 cells, cocultured with BMSC in the presence of ALCAM-Fc chimera fusion protein (AL-Fc, 0.5 μg/ml) for 24 h and the result quantification (right panel). D Hoechst staining of CTR-KD or AL-KD RPMI8226 cells, cocultured with BMSC in the presence of EGFR inhibitor (gefitinib, 200 nΜ) for 24 h and the result quantification (right panel). E Hoechst staining of RPMI8226 cells in the presence of CD6 antibody (10 μg/ml) and the result quantification (right panel). Data are the mean of three independent experiments in three replicates. *p < 0.05.

Fig. 3. ALCAM suppresses the hedgehog pathway activation.

A GSEA analysis showing the hedgehog pathway gene enrichment in SP and MP cells; B Heatmap showing differentially regulated hedgehog pathway gene expression. From left to right are comparisons of CK-KD SP vs. AL-KD SP, CK-KD MP vs. AL-KD MP, CK-KD MP vs. CK-KD SP, and AL-KD MP vs. AL-KD SP. p < 0.05 for all listed genes. C Reverse transcriptional quantitative PCR for hedgehog pathway gene expression in CTR-KD versus AL-KD MM cells. D Hoechst staining of CTR-KD or AL-KD RPMI8226 cells, cocultured with BMSC in the presence of GLI inhibitor (GNAT61, 10 μΜ) and the result quantification (below panel). E Hoechst staining of CTR-KD or AL-KD RPMI8226 cells, cocultured with BMSC or recombinant EGF protein (10 ng/ml) in the presence of SMO inhibitor (cyclopamine, 50 nM) for 24 h and the result quantification (right panel). Data are the mean of three independent experiments in three replicates. *p < 0.05.

Fig. 4. ALCAM regulates myeloma chemoresistant side population in vitro.

A MM cells RPMI8226, either CTR-KD or AL-KD, were treated with melphalan (Mel, 15 μM) or bortezomib (BTZ, 5 nM) for 24 h. The SP cell ratio was examined by Hoechst staining. B The RPMI8226 cells were treated by melphalan as described above. The cell cycle was analyzed after Hoechst staining. C Cell-cycle quantification. D After Hoechst staining, the apoptotic cells were analyzed by annexin V staining. E ALCAM and EGFR expression on MM cells after Mel or BTZ treatment were detected by flow cytometry. MFI mean fluorescence index. F Examination of SP cells after EGFR inhibitor (gefitinib, 200 nM) and melphalan treatment. Data are the mean of three independent experiments in three replicates. *p < 0.05.

Fig. 5. Myeloma side population cells have increased the number of mitochondria.

A Transmission electron microscopy images of RPMI8226 SP and MP cells. B Mitochondria DNA (mtDNA) of RPMI8226 SP and MP cells were examined by qPCR. C Mito-tracker staining of RPMI8226 SP and MP cells. MFI mean fluorescence index. D RPMI8226 MM cells were treated with oligomycin (Oligo, 3 μM) or rotenone (ROT, 1 μM) for 4 h. The SP cell ratio was examined by Hoechst staining. Data are the mean of three independent experiments in three replicates. *p < 0.05.

Fig. 6. EGFR-targeting therapy attenuates side population conferred myeloma chemoresistance in vivo.

A Scheme graph showing the animal study to evaluate the efficacy of the combination therapy (Mel and EGFR inhibitor) in vivo. The mice were treated by intraperitoneal injection of melphalan (60 μg/mouse per time, 4 times within 10 days) or gefitinib (500 μg/mouse per time, 4 times within 10 days), or a combination of both. Each group contained 8 mice. B Tumor-bearing mice were subjected to in vivo bioluminescent imaging (IVIS) before and after treatment. Five out of seven representative results are shown. C The relative luciferase activity of IVIS was calculated. D The tumor-bearing mice were treated twice as described above. Then, mice BM cells were analyzed by Hoechst staining for MM SP in vivo. Two out of three representative results are shown in the left panel, and result quantification is shown in the right panel. E Tumor burden was evaluated by circulating IgG2b. F Treatment efficacy was evaluated by mouse survival. G MM tumor-bearing mice were treated by intraperitoneal injection of PBS, bortezomib (15 μg/mouse per time, 4 times within 10 days), or a combination of bortezomib and gefitinib (500 μg/mouse per time, 4 times within 10 days). The PBS group contained 4 mice; the other treated group each contained 6 mice. Tumor burden was evaluated by circulating IgG2b. H Treatment efficacy was evaluated by mouse survival (*p < 0.05).

Fig. 7. ALCAM expression and stemness myeloma cells.

A ALDEFLUOR analysis of BM cells from non-tumorous donors (n = 5) and MM patients (n = 7). Two representative results are shown. B BM cells from 42 newly diagnosed MM patients were analyzed by flow cytometry for ALCAM expression in CD138⁺ MM cells and ALDEFLUOR staining. Results show 2 representatives of ALCAM-high MM versus ALCAM-low MM, and corresponding ALDEFLUOR staining results. C Graphic model of EGFR-targeting therapy in MM treatment (*p < 0.05).