Cancer-testis antigen MAGEC2 promotes proliferation and resistance to apoptosis in Multiple Myeloma
Nesrine Lajmi
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorTim Luetkens
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorSara Yousef
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorJulia Templin
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorYanran Cao
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorYork Hildebrandt
Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorKatrin Bartels
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorNicolaus Kröger
Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorCorresponding Author
Djordje Atanackovic
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Correspondence: Djordje Atanackovic, Division of Hematology and Hematologic Malignancies, University of Utah/Huntsman Cancer Institute, Room HCI 4265, 2000 Circle of Hope Drive, Salt Lake City, UT 84112, USA.
E-mail: [email protected]
Search for more papers by this authorNesrine Lajmi
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorTim Luetkens
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorSara Yousef
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorJulia Templin
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorYanran Cao
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorYork Hildebrandt
Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorKatrin Bartels
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorNicolaus Kröger
Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Search for more papers by this authorCorresponding Author
Djordje Atanackovic
Hematology and Hematologic Malignancies, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA
Oncology/Haematology/Bone Marrow Transplantation with the section Pneumology, University Medical Centre Hamburg-Eppendorf, Hamburg, Germany
Correspondence: Djordje Atanackovic, Division of Hematology and Hematologic Malignancies, University of Utah/Huntsman Cancer Institute, Room HCI 4265, 2000 Circle of Hope Drive, Salt Lake City, UT 84112, USA.
E-mail: [email protected]
Search for more papers by this authorSummary
Cancer-testis antigens belonging to the MAGE family of genes, such as MAGEC2, are commonly and specifically expressed in Multiple Myeloma (MM) and are associated with a more aggressive clinical course and chemotherapy resistance. MAGEC2 is thought to be an excellent candidate for cancer immunotherapy; however, the biological role of MAGEC2 in MM has remained unclear. We investigated the biological role of MAGEC2 in myeloma cells determining the effect of MAGEC2 knockdown on proliferation and apoptosis. Loss of MAGEC2 resulted in reduced proliferation, viability, and anchorage-independent growth of myeloma cells irrespective of the functional status of TP53 (p53). The anti-proliferative effect of MAGEC2 silencing was due to a decrease of cells in the S phase, cell cycle delay at both G0/G1 and/or G2/M, and an increase in the sub-G0/G1 diploid population related to apoptotic cell death. Importantly, overexpression of short hairpin (sh)RNA-refractory MAGEC2 rescued the anti-proliferative effect of mRNA knockdown and protected cells from apoptotic cell death. Our findings support a TP53-independent role of MAGEC2 in promoting the survival of myeloma cells suggesting that MAGEC2-specific immunotherapies have the potential to eradicate the most malignant cells within the myeloma tumour bulk leading to durable clinical responses.
Supporting Information
| Filename | Description |
|---|---|
| bjh13762-sup-0001-Supinfo.docWord document, 118 KB | Data S1. Supplementary methods. Table S1. Primer sequences, annealing temperatures and product size for PCR amplification. Table S2. shRNA and siRNA sequences and sequence targets. |
| bjh13762-sup-0002-FigS1.tiffTIFF image, 7.2 MB | Fig S1. Analysis of MAGEC2 expression in PBMC from healthy donors. (A) Analysis of MAGEC2 mRNA expression in PBMC by qualitative RT-PCR. (B) Western blot analysis of MAGEC2 protein expression in PBMC from healthy donors and GST-MAGEC2 recombinant protein (Abnova). ACTB served as a loading control. GST-MAGEC2 recombinant protein served as a positive control. (PBMC, peripheral blood mononuclear cells; HD, Healthy donors; Neg Control, Negative control) (C) Analysis of the ectopic expression of MAGEC2 in cell line HEK-293T by western blot. ACTB served as a loading control. IM-9 lysate served as a positive control. |
| bjh13762-sup-0003-FigS2.tiffTIFF image, 2.1 MB | Fig S2. Amplification of TP53 for Sanger sequencing. The mutational status of TP53 in myeloma cell lines MOLP-8, OPM-2, SKO-007, IM-9, U-266, RPMI-8226 and EJM was determined by genomic-based sequencing of 13 amplicons across the entire TP53 coding region, the untranslated exon 1 and the A promoter by designing primers that anneal to sequences located at 20 bp from the ends of each exon. Ethidium bromide stained PCR products are shown of all TP53 Exons and promoter A (PA) amplified using a genomic DNA extracted from MOLP-8 myeloma cell line as a template. |
| bjh13762-sup-0004-FigS3.tiffTIFF image, 18.1 MB | Fig S3. Transient siRNA-mediated silencing of MAGEC2 in myeloma cell line MOLP-8. MOLP-8 (wt-TP53) was transfected with three different siRNAs targeting MAGEC2 mRNA, 2 scrambled siRNA, and FITC-labelled non-specific siRNA oligo. After 24 h, the efficiency of FITC-labelled non-specific siRNA oligo uptake was assessed by (A) phase inverted/fluorescence microscopy and (B) by flow cytometry. (C) MAGEC2 knockdown efficiency was assessed by quantitative RT-PCR at 48 h post transfection. The graph depicts the remaining expression of MAGEC2 mRNA in myeloma cells transfected with MAGEC2 siRNA, normalized to the expression of GAPDH housekeeping gene and relative to the expression of MAGEC2 mRNA in myeloma cells treated only with Lipofectamine 2000 (mock transfection). (D) Western blot analysis of MAGEC2 knockdown kinetics in MOLP-8 was performed with whole cell lysate harvested at 48, 72, 96 and 120 h post transfection. ACTB served as a loading control. |
| bjh13762-sup-0005-FigS4.tiffTIFF image, 974.6 KB | Fig S4. Schematic of the inducible shRNAmir vector. Schematic presentation of Tet-on lentiviral inducible RFP-shRNAmir and MAGEC2∆3′UTR-TurboRFP-shMAGEC2mir used for MAGEC2 silencing and rescue, respectively. |
| bjh13762-sup-0006-Legends.docxWord document, 19.6 KB |
Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
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