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.
Adenoid cystic carcinoma (ACC) is a relentless form of salivary gland cancer, typically treated with surgery and radiation, as these tumors are resistant to chemotherapy. In advanced disease, cisplatin is usually the first treatment option, but clinical response and long-term efficacy are modest at best. Here, we demonstrate that therapeutic inhibition of the MDM2–p53 interaction with a small molecule (MI-773) sensitizes adenoid cystic carcinomas to cisplatin in patient-derived xenograft (PDX) and low passage primary human ACC models. Combination of MI-773 and cisplatin mediates potent and durable tumor regression via p53 reactivation and tumor cell apoptosis. MI-773 reduces the fraction of cancer stem cells, which have been associated with resistance to therapy. Notably, neoadjuvant therapy with MI-773 and cisplatin eliminated recurrences of PDX ACC tumors for at least 300 days. Collectively, these data suggest that patients with ACC might benefit from combination therapy with a MDM2–p53 inhibitor and cisplatin.
Salivary gland tumors are typically not responsive to conventional chemotherapy. Adenoid cystic carcinoma (ACC) is one of the most common malignancies arising from minor and major salivary glands (1). Despite the apparent “benign” behavior characterized by slow tumor growth, ACC is relentless. It is highly destructive to adjacent anatomic sites and exhibits a high frequency of perineural invasion events that leads to severe morbidity, particularly as this tumor grows toward the brain (2). The slow and indolent clinical course often leads to a late diagnosis, when tumor is already at advanced stages and systemic intervention is required to prevent distant metastasis and recurrence after surgical resection. Unfortunately, current standard chemotherapy (e.g., platinum-based drugs) has shown limited long-term efficacy in ACC patients (3–5). The prevalence of recurrence is high mainly due to late hematogenous dissemination of cancer cells primarily to lungs, bone, and liver (4). Novel mechanism-based therapies to overcome drug resistance are desperately needed to enhance the survival and quality of life for patients with ACC.
The mechanisms involved in the resistance of ACC to chemotherapy are poorly understood. The first-line chemotherapy for ACC often includes cisplatin, which is frequently combined with other classes of antineoplastic agents [e.g., 5-fluorouracil (5-FU); refs. 3, 6]. However, data from clinical trials with these drug combinations are not promising, with modest efficacy in ACC (7). The combination of cisplatin with doxorubicin and cyclophosphamide mediated only partial therapeutic responses (8). Another study tested the efficacy of combination cisplatin, 5-FU, and epirubicin in patients with advanced, symptomatic ACC (9). From 8 patients enrolled in this study, one showed partial response to therapy and 5 had stable disease. Besides the low sample size, one important limitation of this study is the total period of evaluation (62.3 months), when it is known that around half of ACCs recur only after 10 years (10). Notably, most ACC tumors express the proto-oncogene c-KIT. However, dasatinib (a c-KIT inhibitor) was not effective in a phase II clinical trial with ACC patients (11). The scarcity of preclinical/clinical evidence and lack of response to conventional drugs in the daily clinical practice is reflected on the most recent guideline from the National Comprehensive Cancer Network (NCCN), in which no specific chemotherapeutic drug/regimen is indicated for the treatment of these patients. To improve the prediction of patient outcomes, NCCN has incorporated nomograms (i.e., statistical model using regression analysis) to guidelines as an alternative method for TNM staging system. Notably, this method was proven to be accurate in the prediction of survival and recurrence in ACC patients (12).
The tumor suppressor p53 is often inactivated in cancer (13). Important functions of wild-type p53 (e.g., cell-cycle control, DNA repair, induction of apoptosis) are regulated by its interaction with murine double minute 2 (MDM2; ref. 14). This protein interaction forms an autoregulatory negative feedback loop, that is, nuclear p53 induces the expression of MDM2, which in turn binds directly to p53, provoking its degradation through the 26S proteasomal pathway. This interaction is important to maintain low cellular levels of p53 in physiologic conditions (14, 15). However, overexpression of MDM2 leads to p53 inactivation, which contributes to tumorigenic processes (16). Of note, MDM2 expression levels have been associated with tumor progression and poor prognosis of ACC patients (17, 18).
Recently, Wang and colleagues have developed a small-molecule inhibitor of the MDM2–p53 interaction (MI-773, patented by Sanofi as SAR405838), pursuing the p53 activation as a new anticancer strategy (19). MI-773 has a high binding affinity to MDM2 (Ki = 0.88 nmol/L), preventing MDM2 interaction with p53. Indeed, MI-773 is capable of wild-type p53 reactivation in cancer cell lines and several preclinical models of cancer, for example, osteosarcoma, leukemia, colon, and prostate cancer (19, 20). Considering the important role of MDM2 in ACC, our group has tested the effect of MI-773 in preclinical models of ACC (21). We observed that therapeutic inhibition of MDM2 with MI-773 restored p53 function, as shown by the activation of its downstream-related protein (i.e., p21). Here, we went a step further and evaluated the effect of MI-773 in combination with cisplatin in preclinical models of ACC. MI-773 and cisplatin drug combination induced robust and durable ACC tumor regression. Furthermore, this therapy significantly decreased the proportion of cancer stem cells (CSC), which has been associated with cisplatin resistance in head and neck tumors (22). Collectively, these results suggest that patients with ACC might benefit from neoadjuvant therapeutic inhibition of the MDM2–p53 interaction combined with conventional chemotherapy with cisplatin.
Materials and Methods
Salivary ACC cells
Primary cell cultures were generated from human tumor specimens and named “University of Michigan Human Adenoid Cystic Carcinoma (UM-HACC)” series, as described previously (21, 23). UM-HACC-2A, UM-HACC-2B, UM-HACC-5, and UM-HACC-6 cells were maintained in salivary gland culture media, that is, high-glucose DMEM (Invitrogen) supplemented with 2 mmol/L l-glutamine (Invitrogen), 1% antibiotic (AAA; Sigma-Aldrich), 10% FBS (Invitrogen), 20 ng/mL EGF (Sigma-Aldrich), 400 ng/mL hydrocortisone (Sigma-Aldrich), 5 μg/mL insulin (Sigma-Aldrich), 50 ng/mL nystatin (Sigma-Aldrich), and 1% amphotericin B (Sigma-Aldrich). Cells were passed using 0.05% trypsin/EDTA (Invitrogen) and used for up to 20 passages. Therefore, these cells are considered here low-passage primary ACC cells (instead of established cell lines). UM-HACC-2A is a low passage cell culture derived from an aggressive primary tumor, and UM-HACC-2B represents ACC cells derived from the lymph node metastasis of the same patient. UM-HACC-5 cells were obtained from a patient that had histologic confirmation of perineural and bone invasion, and UM-HACC-6 cells were derived from a recurrent tumor diagnosed 15 years after initial diagnosis and also presented perineural invasion. All low passage ACC cell cultures used here express wild-type p53 (21).
Studies with patient-derived xenograft model of ACC
Patient-derived xenograft (PDX) models have been increasingly used in translational cancer research (24). Tumor fragments from a PDX model of ACC (UM-PDX-HACC-5; refs. 21, 23) at in vivo passages 5 to 6 were transplanted into the subcutaneous space of the dorsum of SCID mice (CB.17.SCID; Charles River Laboratories). Tumor volume was calculated using the formula: volume (mm3) = L × W2/2 (L, length; W, width). All tumors were surgically retrieved when they reached our UCUCA-approved cut-off volume of 2,000 mm3. The protocol for animal care was reviewed and approved by appropriate University of Michigan institutional committees. Mice transplanted with UM-PDX-HACC-5 tumors were randomly allocated into four groups (n = 5–6), maintaining the average of tumor volume (500 mm3/group). Experimental conditions were as follows: vehicle control (saline, intraperitoneally, weekly); 5 mg/kg cisplatin (intraperitoneally, weekly); 200 mg/kg MI-773 (gavage, weekly); or the combination of 5 mg/kg cisplatin + 200 mg/kg MI-773 regimens. Treatment was administered for 30 days, mice were followed for additional 24 days, and then mice were euthanized and tumors retrieved. To compare weekly versus daily regimens of MI-773 combined with fixed doses of cisplatin, 200 mg/kg MI-773 was divided into equable daily doses (i.e., 28.5 mg/kg MI-773). When tumor reached an average volume of 200 mm3, animals were randomly distributed into the experimental groups (n = 7–8). Treatment was administered for 14 days, and mice were followed for additional 44 days, when experiment was terminated and tumors were collected. To evaluate tumor recurrence, animals were submitted to a neoadjuvant regimen of MI-773. Briefly, mice transplanted with UM-PDX-HACC-5 tumors (average volume of 500 mm3) were divided into two groups (n = 8): vehicle control (polyethylene gycol-200 + d-α-tocopherol polyethylene glycol 1000 succinate; Sigma-Aldrich) or 200 mg/kg MI-773. Initially, animals received either a single dose of MI-773 or vehicle. After 7 days, tumors were surgically removed and incisions were closed with Vetbond (3M). Weekly treatments restarted 7 days after surgery and continued for 30 days. Then, mice were examined twice per week for 300 days for signs of recurrence, as defined as the presence of palpable tumor.
Salivary gland CSCs were identified as ALDHhighCD44high cells, as described previously (25, 26). UM-PDX-HACC-5 tumors were collected, cut in small pieces, and dissociated using the gentleMACS Dissociator Kit (Miltenyi Biotec), incubated in ACK red blood cell lysis buffer (Invitrogen), and filtered through a 40-μm sterile cell strainer. Cells were incubated with 5 μL activated Aldefluor substrate (BAA) or 5 μL aldehyde dehydrogenase (ALDH) inhibitor (DEAB), using the Aldefluor Kit (Stem Cell Technologies) for 40 minutes at 37°C. A monoclonal mouse anti-human CD44 antibody (1:60; APC-cat #559942, PE-cat #550989; BD Pharmingen) was incubated for 30 minutes at 4°C, and human cells were separated from murine cells using anti-HLA-ABC (PE-cat #560168; BD Pharmingen). Viable cells were stained using 7-AAD (cat #00-6993-50; eBioscience). For cell-cycle analysis, 5 × 105 UM-HACC-5 or UM-HACC-6 cells were treated with either vehicle control, MI-773, and/or cisplatin diluted in salivary gland medium for 24 hours. After harvesting, cells were fixed in 70% cold ethanol and exposed to 0.1% sodium citrate, 25 μg/mL propidium iodide (Sigma-Aldrich), 100 μg/mL RNase A, and 0.1% Triton X-100. Flow cytometry (FACSCalibur; BD Biosciences) was carried out to quantify the percentage of cells in each cell-cycle phase (G0–G1, S. and G2–M), as described previously (23). Data were obtained from triplicates and represent at least three independent experiments. Results were analyzed using FlowJo software (FlowJo, LLC).
Cytoplasmic and cellular membrane portions were separated from the nucleus, as described previously (27). Briefly, UM-HACC-5 cells were treated with vehicle control, Cisplatin and/or MI-773 for 24 hours. Then, cells were resuspended in cytoplasmic extraction buffer (Panomics) and incubated for 3 minutes on ice. The suspension was centrifuged at 13,000 rpm for 5 minutes at 4°C. Supernatant was collected as cytoplasmic/cell membrane extract. The remaining pellet was resuspended in nuclear extraction buffer (Panomics) with occasional vortexing during 40 minutes. After centrifugation, the supernatant was collected as nuclear extract.
Protein lysates from UM-PDX-HACC-5 tumors, UM-HACC-5, and UM-HACC-6 cells were prepared using 1% Nonidet P-40 (NP-40) lysis buffer and loaded onto 9% to 12% SDS-PAGE gels. Membranes were incubated with the following primary antibodies overnight at 4°C: mouse anti-human p53 (1:1,000), β-actin conjugated with horseradish peroxidase (HRP; 1:100,000), rabbit anti-hnRNP (1:1,000) (Santa Cruz Biotechnology); hamster anti-human Bcl-2 (1:500), rabbit anti-human phospho-p53 (S392; 1:1,000), p21 (1:2,000), BAX (1:500), PUMA (1:500), cleaved-caspase 9 (1:1,000) (Cell Signaling Technology); mouse anti-human MDM2 (1:500; Thermo Fisher Scientific); rabbit anti-human Bcl-xL (1:500; BD Biosciences), or mouse anti-GAPDH (1:1,000,000; Chemicon). Membranes were exposed to affinity-purified secondary antibodies (1:1,000) conjugated with HRP (The Jackson Laboratory), and immunoreactive proteins were visualized by SuperSignal West Pico chemiluminescent substrate (Thermo Scientific).
Sulforhodamine B (SRB) assays were performed to evaluate the effect of treatment on ACC cell viability, as described previously (23). Here, 2 × 103 UM-HACC-5 and UM-HACC-6 cells per well were exposed to vehicle, MI-773, and/or cisplatin for 24 to 96 hours. After fixation with 10% cold trichloroacetic acid for 1 hour at 4°C, viable cells were stained by addition of 0.4% SRB dye (Sigma-Aldrich) for 30 minutes at room temperature. The excess unbound dye was removed by 1% acetic acid. SRB-stained cells were resolubilized in 10 mmol/L unbuffered Tris base, and plates were read in a microplate reader at 560 nm (GENios, Tecan). Results were normalized by vehicle control and IC50 values were determined. Data were obtained from quadruplicate wells/condition and represent at least three independent experiments.
UM-HACC-5, -6 cells were treated with IC50 concentrations of MI-773, cisplatin, or vehicle control for 24 hours. Cell pellets were resuspended in lysis buffer, and cells were incubated with 1 mmol/L IETD-AFC (caspase-8 substrate; Enzo) or 1 mmol/L LEHD-AFC (caspase-9 substrate; Enzo) for up to 2 hours at 37°C. Samples were analyzed in a microplate reader at 400 nm (GENios).
Data were analyzed by one-way ANOVA, followed by post hoc tests (Tukey) for multiple comparisons or Student t test, when indicated. Regression modeling was performed using mixed effect linear regression to account for repeated measures on each tumor. The tumor volume data was log transformed to account for exponential growth. Model fixed effects included time, cisplatin treatment, and MI-773 treatment, and tumor starting size. Random effects included mouse. We assumed an autoregressive correlation structure where more proximate time values have a higher degree of correlation. When comparing different dosing schemas, each dosing category was included as a different factor level in the regression model. Predictions curves were generated directly from the regression model. Kaplan–Meier graphs were analyzed by the Gehan–Breslow–Wilcoxon test. Failure criterion was a 2-fold increase in tumor volume (for tumor growth analysis) or presence of palpable tumor (for recurrence analysis). Significance level (α) was determined at P < 0.05. Analysis was performed using the “survival” and “gls” packages in software program, version R 3.1.0.
MI-773 sensitizes ACC PDX tumors to cisplatin
To begin to understand the effect of combining MI-773 with cisplatin in vivo, we performed a pilot study to evaluate drug toxicity. Using published studies as reference, we choose weekly doses of 5 mg/kg cisplatin (22) and 200 mg/kg MI-773 (21) for this experiment. Weight loss did not exceed 20% (compared with pretreatment values), which is our UCUCA-approved cutoff for systemic toxicities, when we used single-agent cisplatin or MI-773 or combination of both drugs (Supplementary Fig. S1). To evaluate the effect of MI-773 and/or cisplatin on UM-HACC-PDX-5 tumors, we allowed tumors to grow to an average volume of 500 mm3 and then began treating mice with the same doses mentioned above for 30 days (Fig. 1). We observed that MI-773 as a single agent causes tumor regression, being more effective than single-agent cisplatin (Fig. 1A and C). Cisplatin therapy shows limited therapeutic response, stabilizing the tumor growth, but not causing PDX tumor regression, as reported in humans (6–9). However, combination of MI-773 and cisplatin was more effective than single-agent therapy. There was no tumor rebound upon termination of treatment within the duration of the follow-up in this initial experiment (about 20 days posttreatment; Fig. 1A). Importantly, mice did not show weight loss beyond the 20% cutoff (Fig. 1B). We performed linear mixed effect regression modeling on the tumor volume to account for repeated measures on each tumor and determine the effects of time, cisplatin treatment, MI-773 treatment, and starting tumor size on tumor volume. The rate of tumor growth was significantly less in MI-773 (P = 0.0002) and cisplatin (P = 0.0017) groups when compared with control (Fig. 1C). Western blot analysis from UM-HACC-PDX-5 tumors shows that therapeutic inhibition of MDM2, combined or not with cisplatin, causes the upregulation of p53 and MDM2 expression (Fig. 1D). MI-773–activated p53 is functional in vivo, as confirmed by the upregulation of its downstream protein p21 and the apoptosis effector protein BAX. In contrast, PUMA appears to be primarily regulated by cisplatin, while the prosurvival protein Bcl-2 did not show considerable changes in expression levels upon treatment. Collectively, these in vivo results demonstrate that MI-773 mediates ACC tumor regression in a preclinical model of ACC.
Effect of MI-773 and/or cisplatin on ACC cell proliferation and survival
To evaluate the therapeutic potential of each drug in vitro, low passage primary human ACC cells (UM-HACC-5 and UM-HACC-6) were treated with either a dose range of MI-773 (0.01–10 μmol/L) or cisplatin (0.02–20 μmol/L). Alternatively, cells were treated with a fixed dose of cisplatin (2 μmol/L) together with increasing concentrations of MI-773. The percentage of viable cells was determined by SRB assay. Data were normalized against vehicle control. We observed a dose- and time-dependent cytotoxic response in both ACC primary cells evaluated. IC50 values for single agent were in the low micromolar range, and for both ACC cells, the IC50 for MI-773 was about half concentration as compared with the IC50 for cisplatin (Fig. 2A). Combination of both agents further reduced the IC50 to 0.73 μmol/L (UM-HACC-5) and 3.79 μmol/L (UM-HACC-6) at 72 hours (Fig. 2A). Representative photomicrographs of cells in culture conditions were taken after 72 hours of treatment using intermediate doses of single or combined drugs regimens (1–2 μmol/L; Supplementary Fig. S2B). To better understand the mechanisms associated with the antitumor effect observed in vivo, UM-PDX-HACC-5 tissue slides were stained for in situ TUNEL to reveal cells undergoing apoptosis (Fig. 2B). The percentage of TUNEL-positive cells is significantly higher (P < 0.05) in animals that received either MI-773 or cisplatin (Fig. 2B and C). Combination therapy induced more apoptosis than cisplatin or MI-773 used as a single agent (Fig. 2C). In addition, we evaluated the effect of treatment on cell cycle. UM-HACC-5 or UM-HACC-6 cells were treated for 24 hours using low concentrations of either MI-773 and/or cisplatin (1–2 μmol/L), and the percentage of cells in each cell-cycle phase was determined by flow cytometry (Supplementary Fig. S3). MI-773 causes cell-cycle arrest at the first checkpoint (G1); a similar trend is seen upon drug combination therapy. As previously reported (28), the majority of cells exposed to cisplatin are retained in the S-phase (Fig. 2D). Taken together, these results indicate that antitumor cell effects seen upon treatment with MI-773 and/or cisplatin involve cell-cycle arrest and induction of apoptosis.
Inhibition of the MDM2–p53 interaction with MI-773 activates p53
To better understand the role of MI-773 on p53 activation in ACC, we first performed Western blot analysis for the basal expression of MDM2 and apoptosis-related proteins (i.e., Bcl-xL and Bcl-2) in UM-HACC-2A, UM-HACC-2B, UM-HACC-5, and UM-HACC-6 cells (Fig. 3A). Next, we treated ACC cells with single-agent MI-773 or cisplatin at intermediate doses (1–2 μmol/L) and observed that p53, MDM2, p21, BAX, and PUMA (except for UM-HACC-6 cells) expression is increased with MI-773, while Bcl-2 expression was slightly decreased upon treatment with cisplatin (Fig. 3B). Then, we evaluated more closely the expression of p53 in UM-HACC-PDX-5 tumors from experiment reported in Fig. 1. Tumors treated with either vehicle or cisplatin show nuclear localization of p53. Surprisingly, the administration of MI-773 as a single agent or combined with cisplatin causes a partial translocation of p53 to the cytoplasm (Fig. 3C). To confirm this apparent translocation of p53, we exposed UM-HACC-5 cells to low concentrations of MI-773 and/or cisplatin (0.1–0.2 μmol/L). After 24 hours, we performed a subcellular fractionation assay to separate the nucleus compartment from the cytoplasm/membrane components. Confirming the data obtained by IHC, tumors exposed to MI-773 alone or in combination with cisplatin presented p53 in both nucleus and cytoplasm. MI-773 caused a robust increase in MDM2 expression, which was observed in both the nuclear and the cytoplasmic extract (Fig. 3D). Immunofluorescence further confirmed the cytoplasmic presence of p53 upon treatment with MI-773 (Fig. 3E).
To further understand the effect of MI-773 or cisplatin on ACC cells, we performed a series of dose- and time-dependent experiments (Fig. 4). MI-773 induces activation of phospho-p53 (SER 392), p53, MDM2, p21, BAX, and PUMA in a dose-dependent manner, with visible effects already being observed with 0.1 μmol/L in both cell lines (Fig. 4A). The prosurvival protein Bcl-xL appears to be upregulated by low concentration MI-773 in both ACC cells and went down to baseline levels at 10 μmol/L (Fig. 4A). Cisplatin requires higher doses for the activation of p53, MDM2, p21, and BAX when compared with MI-773 (Fig. 4A and B). The activation of BAX by cisplatin appears to be associated with Bcl-xL downregulation and does not involve PUMA regulation, which differs from MI-773 treatment.
In a time course evaluation with intermediate dose of MI-773 (1 μmol/L), p53 and p21 were constantly activated over 96 hours, while the expression of MDM2 increased with time particularly in UM-HACC-6 cells (Fig. 4C). Upon cisplatin treatment (2 μmol/L), the peak of p53 activation was at 48 hours in UM-HACC-5 cells; the same trend was observed for MDM2. In UM-HACC-6 cells, p53 activation was constant, but MDM2 expression increased over time (Fig. 4D). To better understand the effect of drugs together, we designed an experiment to evaluate the effect of minimal doses of MI-773 and cisplatin on p53 and MDM2. We observed that MI-773 appears to be the dominant factor driving p53 and MDM2 expression when used in combination with cisplatin in the four low passage ACC cell cultures tested here (Fig. 4E). Collectively, these results demonstrate that MI-773 induces p53, MDM2, and p21 expression via a mechanism that is associated with upregulation of Bax and PUMA. To further understand the mechanisms involved in MI-773–induced apoptosis, we performed fluorometric assays. They revealed that caspase-9 (but not caspase-8) is activated upon treatment of ACC cells with MI-773 and/or cisplatin (Fig. 4G). Of note, the activation of caspase-9 in UM-HACC-5 cells treated with MI-773 + cisplatin (120 minutes) is 5.94-fold higher than vehicle control, and in UM-HACC-6, it is 6.76-fold higher than vehicle (Fig. 4G). These data indicate that this drug combination is equally effective in both ACC cell cultures. Western blots were used to verify the data obtained with the fluorometric assay and confirmed that treatment with MI-773 and/or cisplatin induces expression of cleaved (active) caspase-9 (Fig. 4F).
Dosing and frequency of MI-773 determines efficacy
To further explore the antitumor effect of the combined therapy, we designed an independent experiment evaluating weekly versus daily regimens of MI-773 combined with fixed doses of cisplatin. When UM-HACC-PDX-5 tumors reached the average volume of 200 mm3, mice were randomly divided into 5 groups (n = 7–8), and treatment was administered for 14 days (Fig. 5). A shorter treatment period was chosen here to focus the evaluation on the posttreatment period. Here, we observed that single drugs, MI-773 or cisplatin, stabilize tumor volume during treatment, but ACC tumor growth resumed as soon as treatment was discontinued (Fig. 5A). When MI-773 and cisplatin were combined, daily and weekly MI-773 regimens caused tumor regression. Remarkably, weekly MI-773 combined with cisplatin had a much more prolonged antitumor effect than daily MI-773 combined with cisplatin, even though the total amount of MI-773 administered in both groups was exactly the same (Fig. 5A). Importantly, none of the treatment regimens resulted in mouse weight loss beyond the 20% cutoff (Fig. 5B). We again performed a linear mixed effect model to evaluate the tumor growth rate over time, now including each dosing strategy as a different factor level. We performed multivariate regression to determine the differences in growth rates between the different treatment approaches. In a model including initial tumor size, overall growth rate, and cisplatin treatment, treatment with MI-773 weekly (P < 0.0001), but not daily (P = 0.6365), decreased the tumor growth rate (Fig. 5C). In addition, we generated Kaplan–Meier curves using as criterion for “event” a 2-fold increase in tumor volume as compared with pretreatment size. MI-773, cisplatin, and daily doses of MI-773 combined with cisplatin extended time to failure significantly as compared with vehicle control (P < 0.05). Notably, weekly MI-773 combined with cisplatin significantly extended time to failure when compared with daily MI-773 combined with cisplatin (P = 0.0042; Fig. 5D). Scatter plot graph was generated to depict tumor volume after 10 days of treatment (Fig. 5E). After 57 days, tumors were collected and processed for protein analysis. As previously noticed (Fig. 1D), therapeutic inhibition of MDM2 by MI-773, as a single agent or combined with cisplatin, causes upregulation of p53, MDM2, and p21 (Fig. 5F). Both daily and weekly combined treatments cause upregulation of apoptosis-related proteins BAX and PUMA. Taken together, the results suggest that weekly MI-773 combined with cisplatin is the preferable treatment schedule in preclinical models of ACC.
Therapeutic inhibition of the MDM2–p53 interaction reduces the fraction of CSCs and prevents recurrence of PDX ACC tumors
It is known that ALDH activity and CD44 expression can be used to identify CSCs in salivary gland tumors (23–26). To elucidate the impact of MI-773 and/or cisplatin treatment on ACC CSCs, mice harboring UM-HACC-PDX-5 tumors were divided in 4 groups (n = 5) and received MI-773 (200 mg/kg) for one week, combined or not with two single doses of cisplatin (at the first and seventh day; Fig. 6A). Two days after completion of treatment, tumors were retrieved and the proportion of CSCs was analyzed by flow cytometry. Notably, MI-773 alone (or in combination with cisplatin) decreased the fraction of CSCs (ALDHhighCD44high) when compared with cisplatin alone or vehicle control (P < 0.05; Fig. 6B and C). Immunofluorescence for ALDH1 suggested the presence of a larger number of ALDH1-positive cells in the vehicle and cisplatin groups, particularly in close proximity to blood vessels and nerves (Supplementary Fig. S4). These findings suggest the existence of perivascular niches for CSCs in ACC, as it has been described for other tumors (29, 30).
It has been postulated that CSCs play an important role in tumor progression, particularly toward locoregional recurrence and/or metastases (31–33). In an attempt to mimic a clinical trial, we treated mice harboring UM-HACC-PDX-5 tumors (n = 8) with a neoadjuvant regimen of MI-773, followed by complete surgical resection of the tumor (Fig. 6D). Confirming data described above, a single dose of 200 mg/kg MI-773 significantly decreases the proportion of CSCs (Fig. 6E). Upon follow-up for 300 days (approximately half of the lifetime of a mouse), we observed that 5 (out of 8) mice from the control group presented tumor recurrence (Fig. 6F). Remarkably, not a single mouse from the MI-773–treated group presented tumor recurrence during this experimental period (Fig. 6F and G).
ACCs are resistant to all conventional chemotherapeutic drugs available today. As a consequence, treatment for patients with this cancer is largely limited to surgery and radiation. The development of a safe and effective therapy is therefore urgently needed. It is known that MDM2 is overexpressed in ACCs (17, 18). We have recently demonstrated that therapeutic inhibition of the MDM2–p53 interaction with MI-773 mediates short-term antitumor effect in preclinical models of ACC (21). Here, for the first time, we show that MI-773 reduces the fraction of ACC cancer stem cells, sensitizes ACC PDXs to cisplatin, and prevents tumor recurrence when used in a neoadjuvant setting.
Mechanisms associated with cancer progression often involve inactivation in p53 tumor suppressor functions, and MDM2 is a common mechanism to inhibit p53 activity (13–16). We demonstrated here that the reactivation of p53 by MI-773 causes a cell-cycle arrest and induces apoptosis of ACC cells. Interestingly, we observed here a partial translocation of p53 from the nucleus to the cytoplasm. Green and Kroemer (34) discussed the cytoplasmic functions of p53 in mitochondrial outer membrane permeabilization–mediated apoptosis. They propose that PUMA (p53-upregulated modulator of apoptosis), a target of nuclear p53, disrupts the Bcl-xL–p53 interaction in the cytoplasm. Thus, p53 is released and forms a complex with BAX, initiating the caspase-dependent apoptotic pathway. Here, we observed the upregulation of BAX in ACC xenograft tumors treated with MI-773 as a single agent or combined to cisplatin. Cisplatin, in turn, was capable of Bcl-2 downregulation in UM-HACC-5 cells. The complementary mechanisms of action of MI-773 and cisplatin might explain the potentiation of the antitumor effect that was observed here when both drugs are used together in vivo.
It has been shown that the combination of MI-319 (an alternate small-molecule inhibitor of MDM2 from the class as MI-773) with cisplatin suppressed cell growth, colony formation, and induced apoptosis of pancreatic cancer cells (35). Another study showed that combination of cisplatin with Nutlin-3a, a different class of MDM2 inhibitor, enhanced apoptosis and reverted cisplatin resistance in ovarian cancer cells (36). Interestingly, the inhibition of MDM2–p53 interaction was able to radiosensitize tumor cells in a wide range of cancers types (e.g., lung, breast, colorectal, melanoma, and sarcoma; ref. 37). Our work demonstrated the therapeutic benefit of MI-773 and cisplatin combination in ACCs and offered a new hypothesis to explain the antitumor effect of this drug combination, that is, the ablation of CSCs. In addition, we demonstrated here that MI-773 decreases the risk of locoregional recurrence in preclinical models, which is likely associated with its effect on eliminating at least in part the highly tumorigenic CSCs.
The long-term prognosis of patients with ACC is directly affected by late episodes of recurrence and/or metastases (10). In head and neck squamous cell carcinomas, resistance to chemotherapy and recurrence has been associated with CSCs (22, 29, 38). This small population of uniquely tumorigenic cells was also found in salivary gland tumors (25, 26), but their sensitivity to chemotherapy is not understood. Here, we observed that cisplatin did not have a significant effect on the fraction of CSCs in ACC xenograft tumors. In contrast, MI-773 significantly reduced the fraction of CSCs when compared with cisplatin or vehicle control. This finding raises a putative hypothesis to explain the long-term antitumor effect of this drug combination in vivo, that is, MI-773 targets the CSCs (resistant to cisplatin) that have been shown to be mediators of tumor recurrence and/or metastases (33, 33). Indeed, when we exposed mice to a neoadjuvant treatment of MI-773 (i.e., a single dose of MI-773 followed by surgical removal of the tumor, and a subsequent MI-773 therapy for 30 days), we confirmed the reduction of CSCs in the excised tumors. Notably, no recurrence was observed in mice treated with MI-773 even after 300 days of follow-up (about half of the lifetime of mice), while more than half of the mice that received vehicle presented recurrent tumors.
In conclusion, this work demonstrated that therapeutic inhibition of the MDM2–p53 interaction with MI-773 is an effective antitumor strategy that mediates ACC tumor regression, reduces the fraction of CSCs, sensitizes xenograft tumors to cisplatin, and prevents tumor recurrence in preclinical models of ACC. These preclinical data provide the conceptual framework for an early phase I trial testing a combination therapy of MI-773 and cisplatin for treatment of patients with recurrent/metastatic ACC. Once clinical efficacy is established, a step further would be the testing of MI-773 as an adjuvant or neoadjuvant therapy along with surgical resection of the tumor.
Disclosure of Potential Conflicts of Interest
S. Wang reports receiving other commercial research support from and holds ownership interest (including patents) in Ascenta Therapeutics. No potential conflicts of interest were disclosed by the other authors.
Conception and design: F. Nör, G.A. Acasigua, J.E. Nör
Development of methodology: F. Nör, K.A. Warner, G.A. Acasigua, J.E. Nör
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): K.A. Warner, G.A. Acasigua, S.A. Kerk, J.I. Helman
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): F. Nör, G.A. Acasigua, A.T. Pearson, M. Sant'Ana Filho, J.E. Nör
Writing, review, and/or revision of the manuscript: F. Nör, K.A. Warner, G.A. Acasigua, A.T. Pearson, S.A. Kerk, J.I. Helman, M. Sant'Ana Filho, J.E. Nör
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Z. Zhang, G.A. Acasigua
Study supervision: Z. Zhang, M. Sant'Ana Filho, J.E. Nör
Other (provided the MDM2–p53 interaction inhibitor): S. Wang
We thank the patients who kindly provided the tumor specimens used to generate the adenoid cystic carcinoma cells and PDX models that enabled this research project. We also thank the surgeons, nurses, and support staff that enabled the process of tumor specimen collection and processing for use in research.
This work was funded by CNPq (to F. Nör), Adenoid Cystic Carcinoma Research Foundation (AACRF), University of Michigan Head and Neck SPOREP50-CA97248 from the NIH/NCI, and grants R01-DE23220 and R01-DE21139 from the NIH/NIDCR (to J.E. Nör)
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