DREAM Complex Modulates GIST Cell Quiescence and Enhances Imatinib-Induced Apoptosis

1. The DREAM Complex Mediates GIST Cell Quiescence and Is a Novel Therapeutic Target to Enhance Imatinib-Induced Apoptosis Sergei Boichuk, Joshua A. Parry, Kathleen R. Makielski, et al. 2013; 73:5120-5129. Published OnlineFirst June 20, 2013. -Cancer Reasearch 报告人：焦鑫艳 报告时间：2014.9.17

2. Cancer Research (Cancer Res)杂志，为美国癌症研究协会(American Association for Cancer Research，AACR)会刊。1916年创刊，半月刊，Pubmed和SCI收录。 1999-2012 目前中国人在该杂志累计发表343篇。 2001-2012 主要发表包括基础研究、临床前及临床、肿瘤预防及生物治疗在内的肿瘤学原创研究论文和综述文章，为国际肿瘤研究领域引用率最高的杂志之一。 近四年的影响因子：

3. Background 1. The majority of gastrointestinal stromal tumors (GIST), the most common mesenchymal tumor of the gastrointestinal tract , are characterized by oncogenic mutations in the KIT or platelet-derived growth factor receptor-a (PDGFRA) receptor tyrosine kinase. 2. GIST can be successfully treated with the small-molecule kinase inhibitor imatinib mesylate (Gleevec). Complete remissions are rare and patients frequently achieve disease stabilization in the presence of residual tumor masses. 3. Discontinuation of treatment can lead to tumor progression.Residual tumor cells are quiescent， remain viable and able to re-enter the cell-division cycle.

4. 4. This reversible exit from the cell division cycle and entry into G0 has previously been shown to involve the anaphase-promoting complex the APCCDH1 –SKP2–p27 Kip1 signaling axis. APC, together with its activator CDH1, promotes the polyubiquitylation and subsequent degradation of SKP2, a substrate adaptor component of the SCF (SKP1–Cullin–F-box) complex. SKP2 loss results in the accumulation of its target, the CDK inhibitor p27 Kip1, and the reinforcement of a quiescent state. In previous study, we could show that this process is active in imatinib-treated GIST cells.

5. 5. A second major group of proteins that negatively regulate the cell cycle. p130 has been shown to accumulate in G0 and is regulated by SKP2. p130 interacts with E2F4 to repress E2F-dependent gene transcription. This DREAM complex is a multisubunit protein complex in mammalian， consists of DP, RBL2 (p130), E2F4 and the mammalian homologs of the Caenorhabditis elegans (C. elegans) synthetic multivulva class B (synMuvB) core gene products LIN9, LIN37, LIN52, LIN53/RBBP4, and LIN54. Specificity tyrosine-phosphorylation–regulated kinase (DYRK), DYRK1A, mediated pLIN52-Ser28 of the DREAM component and pLIN52-Ser28 was shown to regulate complex formation in G0.

6. 6. Imatinib induces GIST cell quiescence in vitro through the APCCDH1 –SKP2–p27 Kip1 signaling axis. 7. DREAM complex, a multisubunit complex that has recently been identified as an additional key regulator of quiescence. 8. Here, we provide evidence that imatinib induces GIST cell quiescence in vivo and that this process also involves the DREAM complex.

7. Material The human GIST cell line GIST882 (derived from an untreated metastatic GIST) For mouse xenograft models GIST882 cells [carrying a KIT p.K642E (exon 13) mutation)] or tumors originating from the biopsies of two patients [bearing KIT p.V650D (exon 11) or KIT p.A502_Y503dup (exon 9) mutations, respectively] were implanted in both flanks of two mice. Second-passage xenografts were generated by explanting established xenografts and implanting them into the flanks of a second set of mice.

8. Methods 1. Cell culture, inhibitor treatments, and siRNA-transfections 2. Immunologic and cell-staining methods ( Coimmunoprecipitation and immunofluorescence analysis) 3. BrdUrd assay 4. Senescence-associated β-galactosidase activity (~ staining kit) 5. Quantitative real-time reverse transcriptase PCR 6. Cell-cycle analysis (Flow Cytometry) 7. GIST xenograft models

9. Sections 1. Imatinib induces GIST cell quiescence in vivo and in vitro. Fig1. 2 S1-S3 2. The DREAM complex is involved in imatinib-induced quiescence. Fig3 3. The DREAM complex is a modulator of the cellular response to imatinib and is a potential therapeutic target. Fig4. 5 S4

10. Immunofluorescence microscopic analysis Figure 1. Imatinib induces GIST cell quiescence in vivo. the quiescence marker p27 Kip1 Figure S1. Expression levels of its upstream regulator SKP2 do not predict p27Kip1 levels after imatinib therapy. mutation Immunohistochemical analysis

11. BrdUrd incorporation 细胞间期 Cellular proliferation detection of the percentage of cells in S-phase DNA合成前期（G1期） DNA合成期（S期） DNA合成后期（G2期） 细胞分裂 Figure 1. Imatinib induces GIST cell quiescence in vitro. M期为细胞分裂期 G0期：离开细胞周期， 停止细胞分裂。

12. A subset of GIST cells showing morphologic signs of apoptosis during imatinib treatment and cell growth completely recovers after imatinib washout. Figure S2. Imatinib treatment leads to quiescence and not senescence in GIST cells

13. Immunoblot analysis Figure 2. Imatinib-induced GIST cell quiescence does not prevent apoptosis upon imatinib re-challenge. Detection of KIT activation and markers of cell-cycle regulation, markers of apoptosis. Cell-cycle reentry after removal of imatinib Pretreated GIST cells retain their esponsiveness to antineoplastic activities of imatinib as suggested by previous clinical reports.

14. No change in the percentage of senescence-associated β–galactosidase (SA β–gal) positive cells B Senescence-associated marker p16 INK4A (CDKN2A) Figure S3. Imatinib does not induce a senescence phenotype in these cells. Immunofluorescence microscopic staining

15. DREAM complex members: p130 (accumulate in G0), E2F4, and LIN37 Figure 3. The DREAM complex is involved in imatinib-induced quiescence. Enhanced formation of a complex among p130, E2F4, and LIN37 after imatinib treatment of GIST cells. Immunofluorescence microscopic analyses

16. Figure S4. Knockdown of single DREAM complex subunits (p130, E2F4, LIN9, LIN37, or LIN54) did not result in a significant increase of imatinib-induced GIST cell apoptosis E2F4/LIN54 knockdown enhanced GIST cell apoptosis. Inhibition of efficient DREAM complex formation resulted in increased baseline proliferation.

17. Figure 4. LIN52 is activated by imatinib and attenuates its proapoptotic activities. pLIN52-S28 increased. Basal expression of DYRK1A and LIN52 remained unchanged. p-LIN52 and DREAM complex formation were reversible after removal of imatinib. A cell-cycle arrest in G2–M in LIN52-depleted GIST cells. A statistically significant increase of apoptosis.

18. Figure 5. Inhibition of DYRK1A enhances imatinib-induced GIST cell apoptosis. DYRK1A is a protein kinase. DYRK1A inhibitor harmine markers of apoptosis (PARP cleavage, cleaved caspase 3) and quiescence (p130) DAPI stain to morphologically detect apoptotic cells

19. Here, we show that • Imatinib induces GIST cell quiescence in vivo and in vitro. • 2. This process involves the DREAM complex as evidenced by upregulation of p130, increased p130/E2F4/LIN37 complex formation, and enhanced phosphorylation of the DREAM subunit LIN52. • 3. Importantly, inhibition of DREAM complex formation, abrogation of quiescence by siRNA-mediated knock-down of LIN52 or the DYRK1A kinase were both found to significantly increase imatinib-induced GIST cell apoptosis. • 4. Therefore, interference with DREAM-mediated quiescence can enhance imatinib-induced apoptosis and anti-GIST cell activity, which emphasizes the relevance of the DREAM complex as novel drug target for more efficient imatinib responses.

20. 谢 谢！