Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility

<div><p>Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; <i>K</i>_i<0.5 nM; cellular IC₅₀ 2–6 nM). Treatment of tumor cells <i>in vitro</i> with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser₁₀ levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser₁₀). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G₁ cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment. Collectively, this study raises a concern that single agent therapies inhibiting Mps1 will not be well-tolerated clinically but may be when combined with a selective CDK4/6 drug.</p></div

Histopathology of the small intestine of PF-7006-exposed mice.

<p>(A) Vehicle-treatment showing diploid mitotic Figs (arrows) (inset) magnification of the image (n = 3). (B) Representative images of histological segments of the small intestine (duodenum, jejunum, ileum) from PF-7006 treated mice (n = 3) displaying abnormal mitotic Figs (arrows).</p

Mps1 PF-7006 inhibitor-induced cytotoxicity as function of retinoblastoma tumor suppressor protein (Rb1) status in tHMEC cultures co-treated with the CDK4/6 inhibitor palabociclib.

<p>Cells were treated with 1 μM palbociclib for 24 hours, 75 nM PF-7006 for 48 hours, or 24 hours of palbociclib (1 μM) followed by 48 hours of 75 nM PF-7006. (A) Flow cytometry analysis of Rb-competent tHMEC cells co-treated with Mps1 and CDK4/6 inhibitors. (B) Same experimental conditions as (A) applied to Rb-deficient tHMEC cells (tHMEC cells constitutively expressing the Human Papilloma Virus E7 oncogene). (C) Assessment of DNA damage (double-stranded breaks) based on γ-H2AX levels as a function of Rb status in tHMEC cells treated with CDK4/6 and Mps1 inhibitors.</p

<i>In vivo</i> pharmacology of PF-7006 in the HCC1806 triple-negative breast cancer model.

<p>(A) Plasma concentration of PF-7006 following oral dosing of SCID mice (n = 3) to PF-7006 (0.2, 1, 5, 25 mg/kg). Error bars are standard deviations. (B) Modulation of mechanistic marker (pHH3-Ser₁₀) in response to PF-7006 treatment as established by ELISA assay (n = 6). Error bars are standard deviations. (C) Modulation of pHH3-Ser₁₀ in response to PF-7006 treatment based on tumor IHC (n = 3). (D) Tumor growth inhibition from PF-7006 treatment of HCC1806 tumor-bearing mice as a function of dosing and scheduling (n = 12). The following schedules were tested: 0.2 mg/k QD x 14 days (0% TGI), 1 mg/kg QD x 14 days (15% TGI, p = 0.26), 1 mg/kg BID x 14 (12% TGI, p = 0.34), 5 mg/kg Q2D x 7 (40% TGI, p = 9.5x10^-3), and 25 mg/kg Q3D x 5 (59% TGI, p = 4.6x10^-5). Error bars are standard error of the mean. (E) Effect of different doses and schedules of PF-7006 administration on body weights (n = 3). The following schedules were evaluated: 0.2 mg/kg Q2Dx7, 0.2 mg/kg Q2Dx14, 1 mg/kg Q2Dx7, 1 mg/kg QD x14, 5 mg/kg Q2Dx7, 5 mg/kg QD x 14 (significant body weight loss), and 25 mg/kg Q2Dx7 (significant body weight loss). Error bars are standard error of the mean. F. Effect on body weight based on intermittent PF-7006 dosing (25 mg/kg Q3Dx7, n = 3). Upon cessation of dosing, treated animals regained body weight. Error bars are standard error of the mean.</p

Effect of Mps1 inhibitor on duration of mitosis.

<p>Duration of mitosis timed from nuclear envelope breakdown to initiation of cytokinesis in three cell lines exposed to vehicle (0.1% DMSO) or an Mps1 inhibitor (50 nM PF-3837) using live-cell imaging (n = 3). The effect of PF-3837 on mitosis duration for tumor cells are highly-significant for T47D (p = 0.001) and MDA-MB-468 (p = 0.0002).</p

Analysis of cell cycle phenotypes induced by exposure of TNBC, basal-a tumor lines to selective Mps1 inhibitors.

<p>Flow cytometry analysis of the breast tumor lines HCC1806 (panel A) and HCC70 (panel B) exposed to one of two different concentrations of PF-7006 (25, 50 nM) for different treatment intervals in cells at various cell cycle stages. The result of analysis using propidium iodide (PI) staining is displayed.</p