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 11

IGF-1 is a downstream target of H3K27 hypomethylation and a key mediator of CAM-DR.

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IGF-1 is a downstream target of H3K27 hypomethylation and a key mediator...
(A) Chromatin suspensions were prepared from RPMI8226 cells in the experiments described in Figure 2A and immunoprecipitated with anti-H3K27me3 and isotype-matched (IgG) antibodies. The resulting precipitants were subjected to PCR to amplify the promoter regions of the IGF1, BCL2, and IRF4 genes (31–33), using primers listed in Supplemental Table 4. Representative data of 50 cycles are shown. Input indicates that PCR was performed with genomic DNA. (B) RPMI8226 cells were cultured with rIGF-1 at 100 ng/ml or vehicle (control) in the absence or presence of 4-OHCY under stroma-free conditions. Cell viability was determined by the MTT reduction assay after 72 hours (left panel). *P < 0.05 against controls at the same concentrations of 4-OHCY (n = 3). Whole cell lysates were prepared simultaneously and subjected to immunoblotting (right panel). (C) RPMI8226 cells were transduced with either the pLL3.7-sh-control (sh-control) or pLL3.7-sh-IGF-1 (sh-IGF-1) vector, whose efficacy was validated by RT-PCR (Supplemental Figure 7), and cultured in the absence or presence of ADM for 72 hours. Cell proliferation was measured by the MTT reduction assay (left panel). *P < 0.05 against sh-controls at the same concentrations of ADM (n = 3). Whole cell lysates were prepared simultaneously and subjected to immunoblotting (right panel). (D) The role of the IGF-1R/PI3K/Akt pathway in epigenetic regulation of CAM-DR in MM cells. See Results and Discussion for details.