The bromodomain inhibitor OTX015 (MK-8628) exerts anti-tumor activity in triple-negative breast cancer models as single agent and in combination with everolimus

Abstract

Triple-negative breast cancer (TNBC) is an aggressive and heterogeneous subgroup of breast tumors clinically defined by the lack of estrogen, progesterone and HER2 receptors, limiting the use of the targeted therapies employed in other breast malignancies. Recent evidence indicates that c-MYC is a key driver of TNBC. The BET-bromodomain inhibitor OTX015 (MK-8628) has potent antiproliferative activity accompanied by c-MYC down-regulation in several tumor types, and has demonstrated synergism with the mTOR inhibitor everolimus in different models. The aim of this study was to evaluate the anti-tumor activity of OTX015 as single agent and in combination with everolimus in TNBC models. OTX015 was assayed in three human TNBC-derived cell lines, HCC1937, MDA-MB-231 and MDA-MB-468, all showing antiproliferative activity after 72 h (GI50 = 75-650 nM). This was accompanied by cell cycle arrest and decreased expression of cancer stem cells markers. However, c-MYC protein and mRNA levels were only down-regulated in MDA-MB-468 cells. Gene set enrichment analysis showed up-regulation of genes involved in epigenetic control of transcription, chromatin and the cell cycle, and down-regulation of stemness-related genes. In vitro, combination with everolimus was additive in HCC1937 and MDA-MB-231 cells, but antagonistic in MDA-MB-468 cells. In MDA-MB-231 murine xenografts, tumor mass was significantly (p < 0.05) reduced by OTX015 with respect to vehicle-treated animals (best T/C = 40.7%). Although everolimus alone was not active, the combination was more effective than OTX015 alone (best T/C = 20.7%). This work supports current clinical trials with OTX015 in TNBC (NCT02259114).

Keywords: OTX015 (MK-8628); bromodomain inhibitor; everolimus; triple-negative breast cancer.

Conflict of interest statement

CONFLICTS OF INTEREST

Esteban Cvitkovic was founder and CSO of Oncoethix SA, and is CEO of Oncology Therapeutic Development. Lucile Astorgues-Xerri, Elodie Odore, Mohamed Bekradda and María E. Riveiro were or are employees of Oncology Therapeutic Development. Francesco Bertoni and Maurizio D’Incalci received institutional funds from Oncology Therapeutic Development. The other authors disclosed no potential conflicts of interest.

Figures

Figure 1. Characterization of OTX015 antiproliferative activity in three TNBC cell models

(A) Cells were seeded under normoxic and hypoxic conditions and treated with OTX015 for 72 h. The antiproliferative effect was determined by cell counting. The diagonal-line pattern () indicates OTX015 concentrations that decreased cell growth by 50% (GI50). For each treatment, the influence of hypoxia on antiproliferative activity was evaluated with the Student t-test. Significant differences in the OTX015 treatments were determined with a one-way ANOVA test (p < 0.01) followed by an SNK a posteriori test; substantial differences (p < 0.05) are indicated as letters above the bars. (B) Cell lines were treated with OTX015 for 72 h (indicated by the green area) and then cultured in drug-free medium. The Y axis represents the number of cells per well evaluated every 24 h after cell seeding (time 0). Significant differences in the number of control and treated cells at each time point were determined by two-way ANOVA test (p < 0.001) followed by a Bonferroni a posteriori test (*p < 0.05, **p < 0.01, ***p < 0.001). Significant differences at each time point after washout versus 72 h-treated cells were determined with one-way ANOVA test (p < 0.01) followed by an SNK a posteriori test (#p < 0.05). (C) Cells were treated with OTX015 for 24, 48 and 72 h and the latter cells were further cultured without OTX015 for 48 h (drug washout). Significant differences in the percentage of OTX015-treated cells in the G1, S and G2/M phases with respect to untreated controls were evaluated by a one-way ANOVA test (p < 0.01) followed by an SNK a posteriori test. After washout, differences in the percentage of cells in a given cell cycle phase between control and pretreated cells were determined with the Student t-test. Significant differences between treated cells and controls were reported for the G1 phase (*p < 0.05, **p < 0.01), S phase (#p < 0.05, ##p < 0.01) and G2/M phase (×p < 0.05, ××p < 0.01). Each dot or bar and vertical line represents the mean ± SEM, respectively (n ≥ 3).

Figure 2. Baseline and post-OTX015 expression of BRD2/3/4 and c-MYC

Basel expression of BRD2/3/4 and c-MYC in terms of protein (A) and mRNA (B) levels were evaluated in the three cell lines by Western blotting and qRT-PCR, respectively. Protein (C) and mRNA (D) levels were evaluated after 24-, 48- and 72-h OTX015 (650 nM in HCC1937 and MDA-MB-468 cells, and 75 nM in the MDA-MB-231 cell line). Significant differences in mRNA levels were determined by one-way ANOVA test (p < 0.01) followed by an SNK a posteriori test (*p < 0.05, **p < 0.01). Western blots are representative of at least three independent experiments. Bars and vertical lines represent the mean ± SEM, respectively (n = 3).

Figure 3. Gene expression profiling in OTX015-treated TNBC cell models

(A) GEP heat maps in OTX015-treated MDA-MB-231 and MDA-MB-468 cell lines, compared to vehicle-treated control cells. Red and blue indicate up-regulated and down-regulated genes respectively. (B) Venn diagram of 81 and 1286 differentially expressed genes in MDA-MD-231 and MDA-MB-468 OTX015-treated cells, respectively. GSEA analyses showed common signatures enriched, using the complete MSigDB (C), up-regulated and (D), down-regulated) and CMAP databases (E) in OTX015-treated MDA-MB-231 and MDA-MB-468 cell lines. FDR, false discovery rate; NES, normalized enrichment score.

Figure 4. Effect of OTX015 on the expression of cancer stem cell markers in TNBC cell models

Cells were exposed for 24, 48 and 72 h to OTX015 (650 nM for HCC1937 - ■ - and MDA-MB-468 - ■ -, and 75 nM for MDA-MB-231 cells - ■ -) and expression of stemness-related genes was determined by qRT-PCR. Differences in the mRNA levels of OTX015-treated cells vs. controls were determined by a one-way ANOVA test (p < 0.01) followed by an SNK a posteriori test (*p < 0.05, **p < 0.01). Bars and vertical lines represent the mean ± SEM, respectively (n = 3).

Figure 5. Antitumor activity of OTX015 in combination with everolimus in vitro and in MDA-MB-231 xenografts

(A) Antiproliferative effects of concomitant OTX015 and everolimus exposure for 72 h was evaluated with the MTT assay in all three TNBC cell lines. Synergism is defined as a combination index < 0.9; values between 0.9 and 1.10 indicate an additive effect (shown as dotted lines); and values > 1.10, indicate antagonism (n = 3). (B) Antitumor effects of 50 mg/kg OTX015 twice daily (□) and 2 mg/kg everolimus thrice daily (RAD001; ) were evaluated as single agents and in combination () in MDA-MB-231 murine xenografts. Tumor weight was compared between drug regimens at each time point using a two-way ANOVA test (p < 0.001) followed by Bonferroni a posteriori test on log-transformed data. Color-filled symbols indicate significant (p < 0.05) differences between treated vs. vehicle mice (·). Significant differences in tumor mass between the combination versus single agent treatment after drug withdrawal are shown (*p < 0.05, **p < 0.01 and ***p < 0.001). Dotted lines define the treatment period. (C) Animal body weight during treatment (dotted lines) and subsequently. Results represent the mean ± SEM (during treatment, n = 9). Arrows in B and C indicate the change in the everolimus dosing schedule. (D) OTX015 levels in plasma and tissues from OTX015-treated mice, 4 h after the last dose. Variations in OTX015 concentrations detected between plasma, tumor and peritumoral tissue were evaluated by a one-way ANOVA test (p < 0.01) followed by an SNK a posteriori test. Letters above the bars indicate substantial differences (p < 0.05). (E) mRNA levels of c-MYC, BRD2/3/4, and CSC markers were determined by qRT-PCR in MDA-MB-231 tumors after 4-week treatment with OTX015 (■), everolimus (RAD001, ■) or the combination (■). Variations between the experimental groups were evaluated by a one-way ANOVA test (p < 0.001) followed by an SNK a posteriori test. Letters above the bars indicate significant differences (p < 0.05) in mRNA levels among the experimental groups. Bars and vertical lines represent the mean ± SEM, respectively (n = 4).