ABSTRACT
The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2- breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G1 kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.
Subject(s)
Cell Cycle/drug effects , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Neoplasms/drug therapy , Female , Humans , Male , Neoplasms/immunologyABSTRACT
Breast cancer patients with tumors lacking the three diagnostic markers (ER, PR, and HER2) are classified as triple-negative (primarily basal-like) and have poor prognosis because there is no disease-specific therapy available. To address this unmet medical need, gene expression analyses using more than a thousand breast cancer samples were conducted, which identified elevated centromere protein E (CENP-E) expression in the basal-a molecular subtype relative to other subtypes. CENP-E, a mitotic kinesin component of the spindle assembly checkpoint, is shown to be induced in basal-a tumor cell lines by the mitotic spindle inhibitor drug docetaxel. CENP-E knockdown by inducible shRNA reduces basal-a breast cancer cell viability. A potent, selective CENP-E inhibitor (PF-2771) was used to define the contribution of CENP-E motor function to basal-like breast cancer. Mechanistic evaluation of PF-2771 in basal-a tumor cells links CENP-E-dependent molecular events (e.g., phosphorylation of histone H3 Ser-10; phospho-HH3-Ser10) to functional outcomes (e.g., chromosomal congression defects). Across a diverse panel of breast cell lines, CENP-E inhibition by PF-2771 selectively inhibits proliferation of basal breast cancer cell lines relative to premalignant ones and its response correlates with the degree of chromosomal instability. Pharmacokinetic-pharmacodynamic efficacy analysis in a basal-a xenograft tumor model shows that PF-2771 exposure is well correlated with increased phospho-HH3-Ser10 levels and tumor growth regression. Complete tumor regression is observed in a patient-derived, basal-a breast cancer xenograft tumor model treated with PF-2771. Tumor regression is also observed with PF-2771 in a taxane-resistant basal-a model. Taken together, CENP-E may be an effective therapeutic target for patients with triple-negative/basal-a breast cancer.
Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Chromosomal Proteins, Non-Histone/genetics , Glycine/analogs & derivatives , Neoplasms, Basal Cell/metabolism , Triple Negative Breast Neoplasms/metabolism , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Chromosomal Proteins, Non-Histone/metabolism , Female , Gene Expression , Glycine/pharmacology , Humans , Kaplan-Meier Estimate , Mice, SCID , Neoplasms, Basal Cell/drug therapy , Neoplasms, Basal Cell/mortality , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/mortality , Xenograft Model Antitumor AssaysABSTRACT
The efficient transfection of cloned genes into mammalian cells system plays a critical role in the production of large quantities of recombinant proteins (r-proteins). In order to establish a simple and scaleable transient protein production system, we have used a cationic lipid-based transfection reagent-FreeStyle MAX to study transient transfection in serum-free suspension human embryonic kidney (HEK) 293 and Chinese hamster ovary (CHO) cells. We used quantification of green fluorescent protein (GFP) to monitor transfection efficiency and expression of a cloned human IgG antibody to monitor r-protein production. Parameters including transfection reagent concentration, DNA concentration, the time of complex formation, and the cell density at the time of transfection were analyzed and optimized. About 70% GFP-positive cells and 50-80 mg/l of secreted IgG antibody were obtained in both HEK-293 and CHO cells under optimal conditions. Scale-up of the transfection system to 1 l resulted in similar transfection efficiency and protein production. In addition, we evaluated production of therapeutic proteins such as human erythropoietin and human blood coagulation factor IX in both HEK-293 and CHO cells. Our results showed that the higher quantity of protein production was obtained by using optimal transient transfection conditions in serum-free adapted suspension mammalian cells.
