Phosphorylation-mediated EZH2 inactivation promotes drug resistance in multiple myeloma
Jiro Kikuchi, … , Bjarne Bogen, Yusuke Furukawa
Jiro Kikuchi, … , Bjarne Bogen, Yusuke Furukawa
Published October 26, 2015
Citation Information: J Clin Invest. 2015;125(12):4375-4390. https://doi.org/10.1172/JCI80325.
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Research Article Oncology

Phosphorylation-mediated EZH2 inactivation promotes drug resistance in multiple myeloma

Abstract

Alterations in chromatin modifications, such as histone methylation, have been suggested as mediating chemotherapy resistance in several cancer types; therefore, elucidation of the epigenetic mechanisms that underlie drug resistance may greatly contribute to the advancement of cancer therapies. In the present study, we identified histone H3–lysine 27 (H3K27) as a critical residue for epigenetic modification in multiple myeloma. We determined that abrogation of drug-induced H3K27 hypermethylation is associated with cell adhesion–mediated drug resistance (CAM-DR), which is the most important form of drug resistance, using a coculture system to evaluate stroma cell adhesion–dependent alterations in multiple myeloma cells. Cell adhesion counteracted anticancer drug–induced hypermethylation of H3K27 via inactivating phosphorylation of the transcription regulator EZH2 at serine 21, leading to the sustained expression of antiapoptotic genes, including IGF1, B cell CLL/lymphoma 2 (BCL2), and hypoxia inducible factor 1, α subunit (HIF1A). Pharmacological and genetic inhibition of the IGF-1R/PI3K/AKT pathway reversed CAM-DR by promoting EZH2 dephosphorylation and H3K27 hypermethylation both in vitro and in refractory murine myeloma models. Together, our findings identify and characterize an epigenetic mechanism that underlies CAM-DR and suggest that kinase inhibitors to counteract EZH2 phosphorylation should be included in combination chemotherapy to increase therapeutic index.

Authors

Jiro Kikuchi, Daisuke Koyama, Taeko Wada, Tohru Izumi, Peter O. Hofgaard, Bjarne Bogen, Yusuke Furukawa

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Figure 7

EZH2 was phosphorylated at S21 in CD138-positive BM cells from MM patients.

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EZH2 was phosphorylated at S21 in CD138-positive BM cells from MM patien...
(A) Cytospin specimens of MM patient–derived BM mononuclear cells were incubated with anti–phospho-EZH2 (S21), followed by staining with Alexa Fluor 488–conjugated anti-rabbit IgG and PE-conjugated anti-CD138 antibodies. See Supplemental Figure 5A for antibody validation. Nuclei were counterstained with DAPI. Scale bar: 20 μm. Data shown are representative of 3 independent experiments (MM patients nos. 1–3). Arrows indicate cells double-positive for CD138 and p-EZH2 (S21); arrowheads indicate cells double-negative for CD138 and p-EZH2 (S21). (B) Whole cell lysates were prepared from CD138-purified BM mononuclear cells from MM patients (no. 4 to no. 6) for immunoblot analysis of EZH2 phosphorylation. KMS12-BM cells cultured under stroma-free conditions serve as a control for underphosphorylated EZH2 at S21. (C) Representative histograms obtained at flow cytometric sorting of CD138-positive cells from BM mononuclear cells of patient no. 6. Over 80% purity was also yielded in patients no. 4 and no. 5 (data not shown).