Dual TTK/PLK1 inhibition has potent anticancer activity in TNBC as monotherapy and in combination

Abstract

Background: Threonine tyrosine kinase (TTK) and polo-like kinase 1 (PLK1) are common essential kinases that collaborate in activating the spindle assembly checkpoint (SAC) at the kinetochore, ensuring appropriate chromosome alignment and segregation prior to mitotic exit. Targeting of either TTK or PLK1 has been clinically evaluated in cancer patients; however, dual inhibitors have not yet been pursued. Here we present the in vitro and in vivo characterization of a first in class, dual TTK/PLK1 inhibitor (BAL0891).

Methods: Mechanism of action studies utilized biochemical kinase and proteomics-based target-engagement assays. Cellular end-point assays included immunoblot- and flow cytometry-based cell cycle analyses and SAC integrity evaluation using immunoprecipitation and immunofluorescence approaches. Anticancer activity was assessed in vitro using cell growth assays and efficacy was evaluated, alone and in combination with paclitaxel and carboplatin, using mouse models of triple negative breast cancer (TNBC).

Results: BAL0891 elicits a prolonged effect on TTK, with a transient activity on PLK1. This unique profile potentiates SAC disruption, forcing tumor cells to aberrantly exit mitosis with faster kinetics than observed with a TTK-specific inhibitor. Broad anti-proliferative activity was demonstrated across solid tumor cell lines in vitro. Moreover, intermittent intravenous single-agent BAL0891 treatment of the MDA-MB-231 mouse model of TNBC induced profound tumor regressions associated with prolonged TTK and transient PLK1 in-tumor target occupancy. Furthermore, differential tumor responses across a panel of thirteen TNBC patient-derived xenograft models indicated profound anticancer activity in a subset (~40%). Using a flexible dosing approach, pathologically confirmed cures were observed in combination with paclitaxel, whereas synergy with carboplatin was schedule dependent.

Conclusions: Dual TTK/PLK1 inhibition represents a novel approach for the treatment of human cancer, including TNBC patients, with a potential for potent anticancer activity and a favorable therapeutic index. Moreover, combination approaches may provide an avenue to expand responsive patient populations.

Keywords: BAL0891; Mps-1; PLK1; SAC; TTK; mitotic checkpoint inhibitor.

Figure 1

BAL0891 is a dual TTK and PLK1 inhibitor that causes rapid SAC disruption and aberrant mitotic exit. (A) Biochemical properties of BAL0891. (B) Overlay of sensorgrams of single-cycle kinetics experiments with BAL0891, using the kinase domain of TTK and full-length PLK1 on a Biacore T200. (C) TTK and PLK1 drug occupancy in THP-1 cells. Samples were collected 0, 4, 24, 48 and 72 h after 2 h treatment with 50 nM BAL0891 followed by wash-out. % drug-occupied TTK (left) and PLK1 (right) was calculated compared to vehicle controls. (D) % cells in G1, S and G2/M cell cycle phases and with higher ploidy (aneuploid) from flow cytometry profiles of HT29 cells blocked in mitosis with nocodazole (100 ng/mL, 7 h) and treated with BAL0891 (50 nM) for 18 h. (E) Immunoblot of mitotic markers from HT29 cell extracts blocked in mitosis with nocodazole (50 ng/mL, 18 h) and then treated with BAL0891 (50 nM) for the indicated times. GAPDH is loading control. (F) Co-immunoprecipitation assays using HT29 cell extracts, showing the interaction between BubR1 and the indicated SAC proteins. Cells were blocked in mitosis with nocodazole (50 ng/mL, 18 h) and treated for the indicated times with 50 nM BAL0891. An IgG antibody was used as negative control.

Figure 2

Efficient SAC disruption and mitotic exit associated with dual TTK and PLK1 inhibition. (A) Quantification of SAC protein interaction (relative protein pull-down) in HT29 cells blocked in mitosis with nocodazole (50 ng/mL, 18 h) and treated with GI₅₀ BAL0891 or CFI-402257 for the indicated times. Interaction was quantified by dividing the intensity of the prey protein by the bait protein (BubR1) from immunoblot analyses. (B) % cells in G1, S and G2/M cell cycle phases and with higher ploidy (aneuploid) from flow cytometry profiles of HT29 cells blocked in mitosis with nocodazole (NOC, 100 ng/mL, 7 h) and treated with GI₅₀ BAL0891 or CFI-402257 for 18 h. Data are presented as the mean ± SEM; n = 3 independent experiments. Statistics: two-way ANOVA with Tukey post-hoc for multiple comparisons. (C) Immunostaining of CENPC (red), BubR1 (green) and DNA (DAPI, blue) in HT29 cells blocked in mitosis with nocodazole (NOC, 100 ng/mL, 7 h) and then treated with GI₅₀ BAL0891, CFI-402257 and/or onvansertib (ONV) for 1 h. Scale bar: 5 μm. (D) Quantification of BubR1 staining intensity at KTs from 3 independent experiments; 8-19 cells/condition were evaluated in each experiment. BAL0891 (BAL), CFI-402257 (CFI), onvansertib (Onv), nocodazole (Noc). Statistics: Brown-Forsythe and Welch ANOVA tests.

Figure 3

BAL0891 elicits potent, single agent antitumor activity in vivo. (A) MDA-MB-231 tumor-bearing animals treated with vehicle or BAL0891, administered IV QW as indicated. Mean tumor volume ± SEM; n = 8/group. (B) MDA-MB-231 tumor-bearing animals treated with vehicle or BAL0891, administered IV 2QW as indicated. Mean tumor volume ± SEM; n = 8/group. (C) H&E of skin/surrounding tissue from animals 1 and 3, from the site of tumor implantation from tumor-free animals treated with 25 mg/kg BAL0891 IV QW as shown in (A). Pictures show skin, panniculus carnosus (asterisk) and subcutaneous fascia (arrow). Small aggregates of polygonal cells (arrowheads) are present which are most likely consistent with macrophages. (D) Representative graphs of TNBC PDX models with differential responses to BAL0891. Tumor-bearing animals treated with vehicle or BAL0891, IV 2QW as indicated. For model BR5017, dosing was initiated at 10 mg/kg and reduced to 8 mg/kg from day 15. Dashed vertical line indicates end of treatment; n = 4 or 5/group. Non-drug related animal deaths: one in the BAL0891 group (day 33) of model BR5337 and one in the vehicle group (day 54) of model BR5337. Drug-related animal deaths: one in the BAL0891 group (day 8) of model BR5017. (E) TTK and PLK1 drug occupancy in MDA-MB-231 tumors. BAL0891 was dosed at 12.5 mg/kg IV 2QW and tumors were collected for analysis 24, 48 and 96 h after 11 treatments (n = 2-3/time point). % drug-occupied TTK (left) and PLK1 (right) was versus vehicle control samples. (F) TTK drug occupancy in vehicle- or BAL0891-treated tumors obtained from (D) 4 h after the last treatment (n = 4 or 5/group). % drug-occupied TTK was versus vehicle control samples.

Figure 4

Synergistic efficacy of BAL0891 in combination with paclitaxel (PTX) or carboplatin (Carbo). (A) PTX (15 mg/kg) was administered IV QW to BR1282 PDX-bearing mice. BAL0891 (8 mg/kg) was administered IV 2QW or QW (4 h before or after PTX, when administered on the same day). Mean TV ± SEM (left) and mean BW changes in % ± SEM (right) are shown (n = 8/group). Drug-related animal deaths: one in the PTX/BAL0891 2QW combination group (day 21). (B) ΔTV (TV on day 16 - TV on day 0) of vehicle, 8 mg/kg BAL0891 IV 2QW, 15 mg/kg PTX IV QW or a combination of paclitaxel and BAL0891 (PTX; BAL) from the efficacy study in (A). The line represents the mean ± SD. Statistical analysis: one-way-analysis of variance (1WA) with Holm-Sidak post-hoc for group comparisons. (C) Carbo (60 mg/kg) was administered IV QW to SK-OV-3 tumor-bearing mice. BAL0891 (7 mg/kg) was administered IV 2QW (1, 4 or 24 h after carbo, when administered on the same day). Mean TV ± SEM (left) and mean BW changes in % ± SEM (right) are shown (n = 8/group). Drug-related animal deaths: one in the 24 h break combination group (day 8). (D) ΔTV (TV on day 23 - TV on day 0) of vehicle, 7 mg/kg BAL0891 IV 2QW, 60 mg/kg carboplatin IV QW or a combination of carboplatin and BAL0891 (Carbo; BAL) from the efficacy study in (C). The line represents the mean ± SD. Statistical analysis: one-way-analysis of variance (1WA) with Holm-Sidak post-hoc for group comparisons.

Figure 5

SAC deactivation by BAL0891 causes aberrant tumor cell division. TTK and PLK1 collaborate in activating the mitotic spindle assembly checkpoint (SAC) at the kinetochore, ensuring correct chromosome alignment and segregation prior to mitotic exit (SAC ON). BAL0891-mediated prolonged inhibition of TTK combined with a transient effect on PLK1 leads to a rapid disruption of the SAC, leaving tumor cells without adequate time for correct chromosome segregation (BAL0891-induced SAC disruption) contributing to a potent anticancer activity. (Figure created with BioRender.com).