Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jan 15;116(3):1027-1032.
doi: 10.1073/pnas.1818798116. Epub 2018 Dec 31.

Dual inhibition of MDM2 and MDM4 in virus-positive Merkel cell carcinoma enhances the p53 response

Donglim Esther Park  1   2 , Jingwei Cheng  2   3 , Christian Berrios  1   2 , Joan Montero  2   3   4 , Marta Cortés-Cros  5 , Stéphane Ferretti  5 , Reety Arora  2   6 , Michelle L Tillgren  7 , Prafulla C Gokhale  7 , James A DeCaprio  8   2   3
Affiliations

Dual inhibition of MDM2 and MDM4 in virus-positive Merkel cell carcinoma enhances the p53 response

Donglim Esther Park et al. Proc Natl Acad Sci U S A. .

Abstract

Merkel cell polyomavirus (MCV) contributes to approximately 80% of all Merkel cell carcinomas (MCCs), a highly aggressive neuroendocrine carcinoma of the skin. MCV-positive MCC expresses small T antigen (ST) and a truncated form of large T antigen (LT) and usually contains wild-type p53 (TP53) and RB (RB1). In contrast, virus-negative MCC contains inactivating mutations in TP53 and RB1. While the MCV-truncated LT can bind and inhibit RB, it does not bind p53. We report here that MCV LT binds to RB, leading to increased levels of ARF, an inhibitor of MDM2, and activation of p53. However, coexpression of ST reduced p53 activation. MCV ST recruits the MYC homologue MYCL (L-Myc) to the EP400 chromatin remodeler complex and transactivates specific target genes. We observed that depletion of EP400 in MCV-positive MCC cell lines led to increased p53 target gene expression. We suspected that the MCV ST-MYCL-EP400 complex could functionally inactivate p53, but the underlying mechanism was not known. Integrated ChIP and RNA-sequencing analysis following EP400 depletion identified MDM2 as well as CK1α, an activator of MDM4, as target genes of the ST-MYCL-EP400 complex. In addition, MCV-positive MCC cells expressed high levels of MDM4. Combining MDM2 inhibitors with lenalidomide targeting CK1α or an MDM4 inhibitor caused synergistic activation of p53, leading to an apoptotic response in MCV-positive MCC cells and MCC-derived xenografts in mice. These results support dual targeting of MDM2 and MDM4 in virus-positive MCC and other p53 wild-type tumors.

Keywords: MDM2–MDM4; Merkel cell carcinoma; casein kinase 1 alpha; lenalidomide; p53.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: M.C.-C. and S.F. are employed by Novartis Institute for Biomedical Research. R.A. was supported by Wellcome Trust DBT India Alliance Early Career Fellowship IA/E/14/1/501773. A patent was filed with the related work.

Figures

Fig. 1.
Fig. 1.
MCPyV LT activates and ST dampens the p53 response. (A) Inducible expression of truncated tumor isoforms of MCV LT increases ARF and p53 target genes in IMR90 cells. Expression of GFP or LT-L21 and -162 truncated LT was induced with doxycycline (DOX) treatment for 24 h. The LT, ARF, and p53 target gene RNA levels were normalized to those of the GFP-induced cells, whereas the GFP levels were normalized to the LT-L21 samples. Data are shown as mean ± SD. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.00005 (Student t test). (B) IMR90 cells were induced to express GFP, ST, LT-L21, or LT-L21 with ST for 40 h. Lysates were prepared before (−) or after (+) DOX. Activation of p53 response is reflected by increased levels of p53, P-p53, acetyl-lysine 382 p53 (Ac-p53), p21, and cleaved PARP (**). (C) Expression of L21, but not an LT mutant in the LXCXE motif (E216K), activates p53 through inhibiting RB and inducing ARF.
Fig. 2.
Fig. 2.
MDM2 and CK1α are transcriptional targets of the ST–MYCL–EP400 complex. (A) Volcano plot illustrating differentially expressed p53 target genes in MKL-1 MCC cell lines after depletion of EP400 with inducible shRNA relative to control shRNA. Each gene log2 fold change (FC) was plotted against the −log10 P value for statistical significance. Green dots indicate genes that meet the twofold change cutoff, and red dots signify adjusted P < 0.1. (B) RT-qPCR was performed with MKL-1 cells after shRNA was induced for 8 d. Reads were normalized to RPLP0 and uninduced samples. The experiment was performed three times and averaged. Data are shown as mean ± SD. *P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.00005 (Student t test). (C) ChIP with MAX, EP400, ST, and IgG antibodies followed by quantitative PCR (qPCR) of indicated promoters in MKL-1 cells. ChIP-qPCR was performed three times with average percent input shown. (D) Depletion of MCV T antigens causes a reduction in the MDM2, CK1α, and MDM4 levels. MKL-1 cells were transduced with shRNAs targeting ST or ST and LT. (E) ChIP-qPCR for the MDM2 promoter in IMR90 cells in the presence (+) or absence (−) of MCV T antigens. ChIP was performed five times. (F) Nutlin-3 treatment does not elicit p53 response, but MCV T antigens increase levels of MDM2, MDM4, and CK1α in IMR90 cells expressing p53DD. MKL-1 and IMR90-p53DD were treated with nutlin-3 (1 μM) for 24 h.
Fig. 3.
Fig. 3.
MDM4 is overexpressed in MCV-positive MCC. (A) RNA from MCC cell lines and human foreskin fibroblasts (HFF) was harvested for RT-qPCR for MDM4 (total), MDM4-FL (full-length variant), and MDM4-S (short splice variant). MDM4 levels were normalized with the geomean of RPLP0, 18s rRNA, and beta-actin RNA controls. Data are shown as mean ± SD. *P < 0.05 for MDM4-FL (Student t test). (B) Western blot of MCC cell lines and HFF with indicated antibodies. MS-1 and MCC13 overexpress p53 due to inactivating mutations, and MKL-2 and MCC26 do not express detectable levels of p53.
Fig. 4.
Fig. 4.
Inhibition of MDM2 and MDM4 enhances p53 activation in MCC cell lines. (A) Lenalidomide enhances p53 activation by nutlin-3 in MKL-1. MKL-1 cells were treated with nutlin-3 (5 μM), lenalidomide (Len; 10 μM), or both for 40 h. (B) MKL-1 (MCV+ MCC) or UISO (MCV− MCC) cells were treated with nutlin-3 (1 μM) with or without lenalidomide (10 μM) for 24 h. Of note, UISO has less MDM2 and MDM4 proteins than MKL-1. (C) Lenalidomide and, to a lesser degree, pomalidomide, but not thalidomide, cooperated with nutlin-3 to activate p53. MKL-1 cells were treated with nutlin-3 with lenalidomide, pomalidomide (10 μM), or thalidomide (10 μM) for 24 h. (D) Lenalidomide treatment reduced MDM4 binding to p53 and activated MDM2. MKL-1 cells were treated with nutlin-3 (5 μM), lenalidomide (10 μM), or both for 40 h and harvested for immunoprecipitation (IP) with antibodies to MDM4, p53, and CK1α followed by Western blotting. (E) Depletion of CK1α by CRISPR transduction enhanced p53 activation by nutlin-3. MKL-1 cells stably expressing each of two CK1α single-guide RNAs (sgRNAs) were treated with nutlin-3 (1 μM) with or without lenalidomide for 24 h. Lenalidomide further decreased CK1α that sgRNAs did not completely deplete. (F) MDM4 inhibitor SC-24-UR99 (UR99) cooperated with MDM2 inhibitors in activating p53. MKL-1 cells were treated with nutlin-3 (1 μM) or HDM201 (0.1 μM) with or without lenalidomide (1 μM) or UR99 (0.1 μM).
Fig. 5.
Fig. 5.
Inhibition of MDM2 and CK1α-MDM4 synergistically induces cell death by apoptosis. (A) MKL-1 and MS-1 cells were treated with MDM2 inhibitors, nutlin-3, RG7388, or AMG232, and XTT assay was performed after 96 h of treatment. **P < 0.005 (multiple t test). (B) Bliss synergy test displays a strong synergy between nutlin-3 and lenalidomide or SC-24–UR99, but not with thalidomide. (C) BH3 profiling was performed with MKL-1 cells treated with lenalidomide, nutlin-3, or both drugs for 16 h. The experiment was performed three times. Data are shown as mean ± SD. *P < 0.05 (Student t test). (D) MKL-1 MCC xenografts in SCID mice respond to the combinational treatment of HDM201 and lenalidomide. HDM201 (40 mg/kg), lenalidomide (50 mg/kg), or both drugs were administered orally daily, starting when xenograft tumors were 200 mm3. Data are shown as mean ± SEM. *P < 0.05 (multiple t test between HDM201 and combination treatments). #The study was terminated because the tumor volume reached maximum permissible size. (E) MCPyV T antigens sensitize MCC for targeting the p53–MDM2–MDM4 pathway.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Becker JC, et al. Merkel cell carcinoma. Nat Rev Dis Primers. 2017;3:17077. - PMC - PubMed
    1. Paulson KG, et al. Merkel cell carcinoma: Current US incidence and projected increases based on changing demographics. J Am Acad Dermatol. 2017;78:457–463.e2. - PMC - PubMed
    1. Feng H, Shuda M, Chang Y, Moore PS. Clonal integration of a polyomavirus in human Merkel cell carcinoma. Science. 2008;319:1096–1100. - PMC - PubMed
    1. Rodig SJ, et al. Improved detection suggests all Merkel cell carcinomas harbor Merkel polyomavirus. J Clin Invest. 2012;122:4645–4653. - PMC - PubMed
    1. Shuda M, Kwun HJ, Feng H, Chang Y, Moore PS. Human Merkel cell polyomavirus small T antigen is an oncoprotein targeting the 4E-BP1 translation regulator. J Clin Invest. 2011;121:3623–3634. - PMC - PubMed

Publication types

MeSH terms