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. 2016 Jul 15;22(14):3550-9.
doi: 10.1158/1078-0432.CCR-15-1698. Epub 2016 Mar 2.

Targeting MDM2 for Treatment of Adenoid Cystic Carcinoma

Kristy A Warner  1 , Felipe Nör  2 , Gerson A Acasigua  2 , Manoela D Martins  3 , Zhaocheng Zhang  1 , Scott A McLean  4 , Matthew E Spector  4 , Douglas B Chepeha  5 , Joseph Helman  6 , Michael J Wick  7 , Christopher A Moskaluk  8 , Rogerio M Castilho  9 , Alexander T Pearson  10 , Shaomeng Wang  11 , Jacques E Nör  12
Affiliations

Targeting MDM2 for Treatment of Adenoid Cystic Carcinoma

Kristy A Warner et al. Clin Cancer Res. .

Abstract

Purpose: There are no effective treatment options for patients with advanced adenoid cystic carcinoma (ACC). Here, we evaluated the effect of a new small molecule inhibitor of the MDM2-p53 interaction (MI-773) in preclinical models of ACC.

Experimental design: To evaluate the anti-tumor effect of MI-773, we administered it to mice harboring three different patient-derived xenograft (PDX) models of ACC expressing functional p53. The effect of MI-773 on MDM2, p53, phospho-p53, and p21 was examined by Western blots in 5 low passage primary human ACC cell lines and in MI-773-treated PDX tumors.

Results: Single-agent MI-773 caused tumor regression in the 3 PDX models of ACC studied here. For example, we observed a tumor growth inhibition index of 127% in UM-PDX-HACC-5 tumors that was associated with an increase in the fraction of apoptotic cells (P = 0.015). The number of p53-positive cells was increased in MI-773-treated PDX tumors (P < 0.001), with a correspondent shift in p53 localization from the nucleus to the cytoplasm. Western blots demonstrated that MI-773 potently induced expression of p53 and its downstream targets p21, MDM2, and induced phosphorylation of p53 (serine 392) in low passage primary human ACC cells. Notably, MI-773 induced a dose-dependent increase in the fraction of apoptotic ACC cells and in the fraction of cells in the G1 phase of cell cycle (P < 0.05).

Conclusions: Collectively, these data demonstrate that therapeutic inhibition of the MDM2-p53 interaction with MI-773 activates downstream effectors of apoptosis and causes robust tumor regression in preclinical models of ACC. Clin Cancer Res; 22(14); 3550-9. ©2016 AACR.

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Figures

Figure 1
Figure 1
MI-773 induces tumor regression in UM-PDX-HACC-5, a PDX model of ACC. A, graph depicting average tumor volume in mice that received 10, 100 mg/kg MI-773 or vehicle via daily oral gavage for 20 days. Treatment started when tumors were approximately 200 mm3. *P<0.05. B, Kaplan-Meier analysis of time to failure, as defined by doubling tumor volume as compared to pre-treatment volume (n=6–8/group). C, photographs of the mice and tumors immediately after euthanasia. D, graphs depicting actual weight and volume of tumors retrieved from the mice at the termination of the experiment. E, graph depicting mouse weight for the duration of the experiment.
Figure 2
Figure 2
MI-773 induces tumor regression in two additional PDX models of ACC. A,B graphs depicting average tumor volume in mice harboring ACCx6 (A) or ACCx9 (B) that received 100 mg/kg MI-773 or vehicle via daily oral gavage. Treatment started when tumors were approximately 200 mm3. C,D graphs depicting mouse weight for the duration of the experiment.
Figure 3
Figure 3
MI-773 induces tumor cell apoptosis. A, photomicrographs of TUNEL-positive cells (green) and DAPI (blue) in tumors from mice that received vehicle, 10 or 100 mg/kg MI-773. B, graph depicting the percentage of TUNEL-positive cells from tumors represented in panel A. C, graph depicting toxicity of MI-773 in UM-HACC-5 cells in vitro, as determined by SRB assay. D, graph depicting the fraction of apoptotic cells after 72 hours of treatment with MI-773, as determined by the fraction of cells in sub-G0G1 after propidium iodide staining. E, graph depicting the fraction of cells/cell cycle phase after 72 hours of treatment with MI-773, as determined by propidium iodide staining. Different low case letters represent p<0.05.
Figure 4
Figure 4
MI-773 induces p53 expression in ACC tumors in vivo. A, photomicrographs of immunohistochemistry for p53 and cytokeratin 7 in vehicle or 100 mg/kg MI-773 treated tumors. Inserts depict representative cells at high magnification. Please note the shift of p53 from the nucleus (vehicle group) to the cytoplasm (MI-773-treated group). B, graph depicting the percentage of p53-positive cells in ACC tumors represented in panel (A). p<0.05. C, graph depicting the localization of p53 (nucleus or cytoplasm) in tumors retrieved from mice that received vehicle or 100 mg/kg MI-773. Different low case letters represent p<0.05. Scale bars=50 μm. D, Western blot showing p53 and phospho-p53 (Ser392) in whole tumor lysates prepared from mice that received vehicle, 50 or 100 mg/kg MI-773.
Figure 5
Figure 5
Effect of MI-773 on its targets and downstream effectors in ACC tumor cells in vitro. A, Western blot for p53, EGFR and E-Cadherin in a panel of low passage primary human cells (UM-HACC series). Mucoepidermoid carcinoma cells (UM-HMC-3A,-3B) served as controls for p53, EGFR and E-Cadherin. B, Western blots showing levels of p53 over different passages in a panel of primary human ACC cells. C,D, Western blots for MDM2, p53, p21 and phospho-p53 (Ser392) expression upon treatment with increasing concentrations of MI-773 (24 hours) in a panel of low passage UM-HACC cells.
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