Page 133 - D. Cancer biology

Combination treatment with GSK126 and pomalidomide induces B-cell differentiation in EZH2
gain-of-function mutant diffuse large B-cell lymphoma
SungryulPark 1,2# ,Seung-Hyun Jo 1,2# , Jong-Hwan Kim 3 , Seon-Young Kim 2,3 , Jong Yeon Hwang 4,5 , Jong Soon Kang 6 ,
, Sung Goo Park 1,2 , Byoung Chul Park 1,2* , SunhongKim 1,2* , and Jeong-HoonKim 1,2*
1 Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
2 KRIBB School of Bioscience, Korea University of Science and Technology, Daejeon 34113, Republic of Korea
3 Personalized Genomic Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, 34141, Republic of Korea
4 Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 305-606, Republic of Korea
5 Medicinal Chemistry and Pharmacology, Korea University of Science and Technology, Daejeon 34113, Republic of Korea
6 Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea
BACKGROUND
Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid neoplasm worldwide. Gene expression profiling has led to the identification
of germinal center B-cell–like (GCB) and activated B-cell–like (ABC) subgroups of DLBCL according to cell origin. Genetic analyses have revealed
recurrent alterations in EZH2, a catalytic component of the Polycomb Repressive Complex 2 (PRC2) complex. In particular, gain-of-function
mutations in its SET domain, including tyrosine 641 (Y641), account for 22% GCB DLBCL, supporting the utility of EZH2 intervention for treatment
of this subtype. PRC2 plays essential roles in silencing of genes involved in cellular differentiation and maintenance of stem cells. The hyperactive
EZH2 Y646 mutant commonly identified in DLBCL induces an aberrant increase in the repressive histone marker, H3K27me3. The potent EZH2
inhibitor, GSK126, has been shown to inhibit H3K27me3 in EZH2 Y641/A677 mutant lymphoma in a dose-dependent manner.
Immunomodulatory drugs (IMiDs), including thalidomide, lenalidomide, and pomalidomide, possess anti-myeloma, anti-inflammatory, and anti-
proliferative properties. Once IMiDs engage the cellular receptor cereblon (CRBN), a ubiquitin E3 ligase, the CRL4 CRBN complex promotes
degradation of neosubstrates.
In this study, we examined the potential cooperativity between PRC2 inhibitors and pomalidomide, with a view to their combined administration as
anti-cancer therapy. PRC2 inhibitors and pomalidomide synergistically and selectively inhibited proliferation of EZH2 mutant DLBCL cells.
RESULTS

▼ Figure 1. Pomalidomide enhances the cytotoxic effect of GSK126 in mutant EZH2 DLBCL cells
(A) Annexin V-FITC (Annexin V)/propidium iodide (PI) staining of SU-DHL6 cells treated with DMSO ,pomalidomide, GSK126 or pomalidomide and GSK126
combination (P+G). (B) Effects of pomalidomide and GSK126 on cell viability. SU-DHL6 cells were treated with the indicated concentrations of GSK126 and
pomalidomide for six days. Combination Index (CI) values obtained for WSU-DLCL2 treated with different concentrations of pomalidomide in combination with
GSK126 (1 μM) (* p-value <0.05, ** p-value <0.005, Student's t-test).
▼ Figure 2. EZH2 mediated synergistic anti-proliferative effect is independent to the lysine methyltransferase (KMTase) activity and is dependent on CRBN
(A) Effects of EZH2 knockdown and pomalidomide on viability of SU-DHL6 cells. (B) WSU-DLCL2 and SU-DHL6 cells were treated with GSK126 (GSK, 1 μM),
pomalidomide (Pom, 1 μM), or both for 48 h, and cell lysates analyzed via western blot using the indicated antibodies. (C) Effects of pomalidomide and GSK126 on
viability of shControl and shCRBN WSU-DLCL2. Cells were treated with the indicated concentrations of GSK126 and pomalidomide (Pom) for 6 days.
▼ Figure 3. Comparative analysis of transcriptomic changes in the GCB-DLBCL by treatment with POM and GSK drugs by RNA-seq.
(A) Heatmap depicting differentially expressed genes in WSU-DLCL2 combination (P+G). Genes were hierarchically clustered (rows). (B) Genes essential for plasma
cell differentiation were upregulated to a more significant extent in P+G than other groups. © RT-qPCR analysis of mRNA levels of genes involved in plasma cell
differentiation. (D) WSU-DLCL2 and SU-DHL6 cells were treated with pomalidomide (Pom, 1 μM), GSK126 (1 μM), and P+G for 48 h. Cell lysates were analyzed via
western blot using the indicated antibodies.
▼ Figure 4. GSK126 and pomalidomide synergistically inhibit tumor growth in vivo
Specific pathogen-free CB17/SCID mice (n=6) were implanted subcutaneously with WSU-DLCL2 cells. (A) Tumor volumes were measured three times a week for 24
days . (B) Tumor weights were measured at the end of the experimental period. (C) Representative H&E staining (top) and Ki-67 (D) immunostaining (bottom) of
xenograft tumors (scale bar, 50 μm).Representative photographs of IRF4 immunostaining (scale bar, 100 μm).

Figure 1 Figure 3 Figure 4

Figure 2

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