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. 2017 Feb 15;23(4):1036-1048.
doi: 10.1158/1078-0432.CCR-16-1235. Epub 2016 Aug 22.

Therapeutic Inhibition of the MDM2-p53 Interaction Prevents Recurrence of Adenoid Cystic Carcinomas

Felipe Nör  1   2 , Kristy A Warner  1 , Zhaocheng Zhang  1 , Gerson A Acasigua  1   2 , Alexander T Pearson  1   3 , Samuel A Kerk  1 , Joseph I Helman  4   5 , Manoel Sant'Ana Filho  2 , Shaomeng Wang  3   5   6   7 , Jacques E Nör  8   5   9   10
Affiliations

Therapeutic Inhibition of the MDM2-p53 Interaction Prevents Recurrence of Adenoid Cystic Carcinomas

Felipe Nör et al. Clin Cancer Res. .

Abstract

Purpose: Conventional chemotherapy has modest efficacy in advanced adenoid cystic carcinomas (ACC). Tumor recurrence is a major challenge in the management of ACC patients. Here, we evaluated the antitumor effect of a novel small-molecule inhibitor of the MDM2-p53 interaction (MI-773) combined with cisplatin in patient-derived xenograft (PDX) ACC tumors.Experimental Design: Therapeutic strategies with MI-773 and/or cisplatin were evaluated in SCID mice harboring PDX ACC tumors (UM-PDX-HACC-5) and in low passage primary human ACC cells (UM-HACC-2A, -2B, -5, -6) in vitro The effect of therapy on the fraction of cancer stem cells (CSC) was determined by flow cytometry for ALDH activity and CD44 expression.Results: Combined therapy with MI-773 with cisplatin caused p53 activation, induction of apoptosis, and regression of ACC PDX tumors. Western blots revealed induction of MDM2, p53 and downstream p21 expression, and regulation of apoptosis-related proteins PUMA, BAX, Bcl-2, Bcl-xL, and active caspase-9 upon MI-773 treatment. Both single-agent MI-773 and MI-773 combined with cisplatin decreased the fraction of CSCs in PDX ACC tumors. Notably, neoadjuvant MI-773 and surgery eliminated tumor recurrences during a postsurgical follow-up of more than 300 days. In contrast, 62.5% of mice that received vehicle control presented with palpable tumor recurrences within this time period (P = 0.0097).Conclusions: Collectively, these data demonstrate that therapeutic inhibition of MDM2-p53 interaction by MI-773 decreased the CSC fraction, sensitized ACC xenograft tumors to cisplatin, and eliminated tumor recurrence. These results suggest that patients with ACC might benefit from the therapeutic inhibition of the MDM2-p53 interaction. Clin Cancer Res; 23(4); 1036-48. ©2016 AACR.

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Figures

Figure 1
Figure 1
Effect of MI-773 and/or Cisplatin in a preclinical model of adenoid cystic carcinoma. UM-PDX-HACC-5 tumors were transplanted in immunodeficient mice. When tumors reached 500 mm3, animals were randomly allocated into four different treatment regimens, as follows: 5 mg/kg saline (vehicle control), 200 mg/kg MI-773, 5 mg/kg Cisplatin, or 200 mg/kg MI-773 combined with 5 mg/kg Cisplatin weekly. A, Graph depicting tumor volume during treatment (tx) and follow up (23 days) periods. Cisplatin stabilized tumor growth, while MI-773 decreases tumor volume compared with pre-treatment values. MI-773 combined with Cisplatin ablates tumor in the majority of animals and prevents tumor regrowth after treatment. B, Graph depicting mouse weight during the experimental period. Data were normalized against pre-treatment weight. C, Graph depicting a linear regression model using repeated measures for each tumor over time. MI-773 and/or Cisplatin significantly decrease the tumor growth rate when compared to vehicle control. D, Western blot for p53, MDM2, p21, BAX, PUMA and Bcl-2 in UM-PDX-HACC-5 tumors treated with either MI-773 and/or Cisplatin as compared to vehicle control. Tumors were harvested 23 days after last administration of drugs.
Figure 2
Figure 2
Effect of MI-773 and/or Cisplatin on proliferation and survival of adenoid cystic carcinoma cells. A, Graphs depicting time- and dose-dependence assays for the effect of MI-773 and/or Cisplatin on the viability of UM-HACC-5 and -6 cells, as determined by SBR assay. Data were normalized against vehicle control and represent at least three independent experiments, done in quadruplicate wells/condition. B, Representative photomicrographs of UM-PDX-HACC-5 tumor histological sections stained for TUNEL (apoptotic cells, green) and DAPI (nuclei, blue) from mice treated either with vehicle control, MI-773 and/or Cisplatin (400×). C, Graph depicting the percentage of TUNEL-positive cells in 10 random fields per tumor (n=4/group). Different low case letters (a,b,c) indicate statistical difference (P<0.05). D, Graphs depicting the effect of the drugs on cell cycle. UM-HACC-5 and -6 cells were exposed to MI-773 and/or Cisplatin for 24 hours. The percentage of cells in each cell cycle phase was determined by propidium iodide staining followed by flow cytometry.
Figure 3
Figure 3
Treatment with MI-773 cause changes in p53 status. A, Western blot showing basal levels of proteins p53, MDM2, Bcl-xL and Bcl-2 in UM-HACC-2A, -2B, -5 and -6 cells. B, Western blots for p53, MDM2, p21, PUMA, BAX and Bcl-2 protein expression in UM-HACC cells exposed to pre-determined IC50 values of MI-773, Cisplatin or vehicle control, for 72 hours. C, Representative photomicrographs of UM-PDX-HACC-5 histological sections stained for p53 (brown) and counterstained with Hematoxylin (200×). Nuclear p53 is observed in mice treated with vehicle control or Cisplatin, while both nuclear and cytoplasmic expression of p53 is evidenced in animals exposed to MI-773 as single agent or combined with Cisplatin (details). D, Western blot for p53 and MDM2 expression in UM-HACC-5 cells exposed to MI-773 (0.1 µg/mL) and/or Cisplatin (0.2 µg/mL) and submitted to a sub-cellular fractionation assay, in order to evaluate the nucleus and cytoplasm compartments in separate. E, Representative photomicrographs obtained by immunofluorescence for p53 (green), which is located in the cytoplasm (detail) of UM-PDX-HACC-5 cells treated with MI-773 as single drug or combined with Cisplatin (400×). Scale bar represents 50 µm.
Figure 4
Figure 4
MI-773 induces apoptosis through PUMA/BAX activation. A and B, Western blots for phosphorylated and total p53, MDM2, p21, BAX, PUMA and Bcl-xL in UM-HACC cells exposed to increasing concentrations of MI-773 (0.01 – 10 µM) or Cisplatin (0.02 – 20 µM), respectively. C and D, Western blots for p53, MDM2 and p21 in UM-HACC cells treated with fixed doses of MI-773 (1 µM) or Cisplatin (2 µM) for 24–96 hours, respectively. E, Western blots for p53 and MDM2 in UM-HACC cells upon treatment with lower doses of MI-773 (0.01 – 0.1 µM) and Cisplatin (0.02 – 0.2 µM) for 24 hours. F, Western blots for cleaved Caspase-9 in UM-HACC-5 and -6 cells exposed to MI-773 and/or Cisplatin for 24 hours. G, Graphs depicting activity of Caspase-8 or Caspase-9 in UM-HACC-5 and -6 cells after 24 hours of treatment with MI-773 and/or Cisplatin, as determined by fluorometric assays. Caspase activity was measured every 20 minutes for a period of 2 hours.
Figure 5
Figure 5
Effect of MI-773 dosing on long-term efficacy. UM-PDX-HACC-5 tumors were transplanted in immunodeficient mice. When tumors reached 200 mm3, animals were randomly allocated into five different treatment regimens, as follows: 5 mg/kg saline (vehicle control), 200 mg/kg MI-773 (weekly), 5 mg/kg Cisplatin, weekly doses of MI-773 (200 mg/kg) combined with 5 mg/kg Cisplatin or daily doses of MI-773 (28.5 mg/kg) combined with 5 mg/kg Cisplatin. A, Graph depicting tumor volume during treatment (tx) and follow up (43 days) periods. Weekly regimens of MI-773 combined with Cisplatin reduce tumors in size and prevent tumor regrowth compared to the other experimental groups or vehicle control. B, Graph depicting mouse weight during the experimental period. Data were normalized against pre-treatment weight. C, Graph depicting a linear regression model using repeated measures for each tumor over time. Weekly regimens of MI-773 combined with Cisplatin significantly decrease the tumor growth rate when compared with daily regimens of MI-773 + Cisplatin or single drug groups (P<0.001). D, Kaplan-Meier analysis using a criterion for failure a 2-fold increase in tumor volume as compared to pre-treatment volume. MI-773, Cisplatin or daily MI-773 combined with Cisplatin extended time to failure significantly as compared to vehicle control (P<0.05). Graph is depicting the statistical difference between daily and weekly regimens of MI-773 in combination with Cisplatin (P=0.0042). E, Graph depicting the volume of each individual xenograft tumor at the 10th post-treatment day. Different low-case letters indicate statistical difference (P<0.05). F, Western blot for p53, MDM2, p21, BAX, PUMA and Bcl-2 in UM-PDX-HACC-5 tumors treated with either MI-773 or Cisplatin as single agents, weekly or daily regimens of MI-773 combined with Cisplatin, or vehicle control. Tumors were harvested 43 days after last administration of drugs.
Figure 6
Figure 6
MI-773 reduces the fraction of cancer stem cells and prevents recurrence in adenoid cystic carcinoma xenograft tumors. A, Timeline showing the experimental design. B, Graph depicting the fraction of cancer stem cells (ALDHhighCD44high) identify by flow cytometry in xenograft tumors treated with 5 mg/kg Cisplatin and/or 200 mg/kg MI-773. Different low case letters (a,b) indicate significant differences among groups (P<0.05). C, Graphs depicting the flow cytometry gates for the percentage of cancer stem cells (Q2) in UM-PDX-HACC-5 tumors treated with Cisplatin and/or MI-773. DEAB is showing the control of the reaction. D, Timeline showing the neoadjuvant treatment design. E, Graph depicting the fraction of cancer stem cells (ALDHhighCD44high) identify by flow cytometry in UM-PDX-HACC-5 tumors treated with MI-773 or vehicle control. F, Macroscopic view of mice harboring recurrent tumors or tumor-free animals at the end of the experiment. G, Kaplan-Meier curve depicting recurrence-free survival in mice treated either with 200 mg/kg MI-773 or vehicle control. Recurrence was defined as the presence of a palpable tumor.
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