Preclinical and clinical studies of gamma secretase inhibitors with docetaxel on human breast tumors

Abstract

Purpose: Accumulating evidence supports the existence of breast cancer stem cells (BCSC), which are characterized by their capacity to self-renew and divide indefinitely and resistance to conventional therapies. The Notch pathway is important for stem cell renewal and is a potential target for BCSC-directed therapy.

Experimental design: Using human breast tumorgraft studies, we evaluated the impact of gamma secretase inhibitors (GSI) on the BCSC population and the efficacy of combining GSI with docetaxel treatment. The mouse experimental therapy paralleled a concurrent clinical trial in patients with advanced breast cancer, designed to determine the maximum-tolerated dose of the GSI, MK-0752, administered sequentially with docetaxel, and to evaluate BCSC markers in serial tumor biopsies.

Results: Treatment with GSI reduced BCSCs in MC1 and BCM-2147 tumorgrafts by inhibition of the Notch pathway. GSI enhanced the efficacy of docetaxel in preclinical studies. In the clinical trial, 30 patients with advanced breast cancer were treated with escalating doses of MK-0752 plus docetaxel. Clinically, meaningful doses of both drugs were possible with manageable toxicity and preliminary evidence of efficacy. A decrease in CD44(+)/CD24(-), ALDH(+), and mammosphere-forming efficiency were observed in tumors of patients undergoing serial biopsies.

Conclusions: These preclinical data show that pharmacologic inhibition of the Notch pathway can reduce BCSCs in breast tumorgraft models. The clinical trial shows feasibility of combination GSI and chemotherapy, and together these results encourage further study of Notch pathway inhibitors in combination with chemotherapy in breast cancer.

Figure 1. Treatment with GSI reduced the BCSC population in patient-derived breast tumorgrafts

Mice with tumors were treated for 3 days with GSI (100 mg/kg) or vehicle, and then tumors were collected 72 hours after the last treatment. A. Treatment with GSI reduced mammosphere forming efficiency (MSFE). Tumors were treated in vivo and subsequently plated under MS conditions. To determine secondary MSFE, primary MS were collected, dissociated, and replated under MS conditions. The Mann-Whitney two-tailed t-test was used to calculate p-values. Top figure, each symbol represents an individual BCM-2147 tumor. Middle panel, MC1 tumors cells were pooled for each treatment group. Each symbol represents a well of MS. Bottom panel, MC1 secondary MSFE. B and C. Treatment with GSI reduced BCSC populations according to flow cytometric analysis of BCSC markers, CD44⁺/CD24⁻ and ALDH+. Treated tumors were dissociated, incubated with antibodies and/or Aldefluor reagents and analyzed by flow cytometry. The Mann-Whitney two-tailed t-test was used to calculate p-values. B. Left column contains representative flow cytometry images. Right column, MC1 CD44/CD24 data (Vehicle n=8, GSI n= 6). C. Left column contains representative flow cytometry images. Top graph MC1 ALDH+ data (Vehicle n=3, GSI n=4). Bottom graph, BCM-2147 ALDH+ data (Vehicle n=9, GSI n=10).

Figure 2. GSI inhibited the Notch pathway

A. Western blot analysis confirmed inhibition the Notch Intracellular Domain (NICD) cleavage. Western blots were incubated with primary antibody against Notch1 or Beta-Actin. Cropped versions of the blots are shown. Image Quant LAS 4000 (GE Healthcare) hardware and software were used to capture multiple digital images over time so that band intensity could be quantitated in the linear dynamic range. The bar graph shows quantification of band intensity to determine NICD relative to Beta-Actin expression. Top panel, MC1; bottom panel, BCM-2147. B. GSI suppressed Notch pathway targets in MC1 tumors. Mice were treated with GSI (n=4) or vehicle (n=4) for two days before collection of tumor tissue on the third day. RTqPCR was performed using ABI Taqman Probes and Primer sets on the ABI 7900HT FAST instrument. Relative gene expression was calculated using the Relative Quantification (RQ) Method, comparing gene expression to 18S ribosomal RNA for each gene. Data is presented relative to the vehicle control. C. GSI suppressed the Notch pathway in the BCSC population. Left panel, relative Hes1 expression in whole tumor (n=3 per group). Right panel, relative Hes1 expression in sorted ALDH+ cells from BCM-2147 tumors (n=2 per group).

Figure 3. Treatment with GSI enhanced efficacy of docetaxel and reduced BCSCs

Mice bearing MC1 or BCM-2147 tumors were stratified by tumor size to treatment groups: docetaxel (10 mg/kg, gray arrows), GSI (100 mg/kg, black arrows), or a combination of both and compared to animals treated with vehicle alone. The tumor growth in different groups of treatment (5 mice per group in A, 6-8 mice per group in B, and 8 mice per group in C) is shown as tumor weight in grams over time or tumor volume fold change. A. The combination of GSI and docetaxel prevented tumor growth more than the single agents. B. GSI alone and with docetaxel reduced tumor growth compared to vehicle [P<0.030 (coefficient = −0.132, 95% confidence interval = −0.252, −0.012); P<0.008 (coefficient = −0.287, 95% confidence interval = −0.498, −0.075, respectively)]. The * indicate statistical differences compared to vehicle. Bottom panel, GSI alone reduced mammosphere forming efficiency (MSFE) compared to vehicle- and docetaxel-treated tumors. Tumors were treated in vivo and subsequently plated under MS conditions. Each symbol represents a well of MS. Square root transformed data were analyzed by with One-Way ANOVA with Bonferonni multiple comparison post test (P<0.05). C. Regimens proposed to be used in human patients were used to assess efficacy of treatment. The difference in tumor growth between mice treated with docetaxel alone and combination of GSI and docetaxel at 3 or 5 days interval is significant (P< 0.01 and 0.05 respectively). GSI alone slows tumor growth significantly compared to control (P<0.03). There was no significant difference between administration of docetaxel 3 days after GSI compared to 5 days after GSI (P<0.09).

Figure 4. Treatment in human clinical trials reduced BCSCs in some patients after multiple rounds of treatment

A. Clinical Trial Design. B. Tumor size of patients that received biopsies for BCSC. Each patient is represented in the same color line across all graphs in B, C, and D. C. Flow cytometry data for CD44⁺/CD24⁻ at baseline, post cycle 1, post cycle 3, and end of treatment (EOT). Note pleural effusate was collected for Pt#9 rather than biopsies, and the line representing Pt#9 is discontinuous based on the break in the y-axis to accommodate larger numbers. D. Flow cytometry data for aldehyde dehydrogenase activity (ALDH⁺). Note: pleural effusate was collected for Pt#9 rather than biopsies, and the line representing Pt#9 is discontinuous based on the break in the y-axis to accommodate larger numbers. E. Patient MSFE decreased after GSI treatment in combination with docetaxel. Pt#9 tumor cells were collected from pleural effusate at baseline and after 4 treatment cycles. Lineage negative tumor cells (<1% of total cells) were sorted into non-adherent plates with mammosphere media for evaluation of MSFE. Each point represents an individual well. MSFE was not evaluated after cycle 2 or cycle 6.