ABSTRACT
Cyclin E is a regulatory subunit of CDK2 that mediates S phase entry and progression. Cleavage of full-length cyclin E (FL-cycE) to low molecular weight isoforms (LMW-E) dramatically alters the substrate specificity, promoting G1/S cell cycle transition and accelerating mitotic exit. Approximately 70% of triple-negative breast cancers (TNBC) express LMW-E, which correlates with poor prognosis. PKMYT1 also plays an important role in mitosis by inhibiting CDK1 to block premature mitotic entry, suggesting it could be a therapeutic target in TNBC expressing LMW-E. Here, analysis of TNBC patient tumor samples revealed that co-expression of LMW-E and PKMYT1-catalyzed CDK1 phosphorylation predicted poor response to neoadjuvant chemotherapy. Compared to FL-cycE, LMW-E specifically upregulated PKMYT1 expression and protein stability, elevating CDK1 phosphorylation. Inhibiting PKMYT1 with the selective inhibitor RP-6306 (lunresertib) elicited LMW-E dependent antitumor effects, accelerating premature mitotic entry, inhibiting replication fork restart, and enhancing DNA damage, chromosomal breaks, apoptosis, and replication stress. Importantly, TNBC cell line xenografts expressing LMW-E showed greater sensitivity to RP-6306 than tumors with empty vector or FL-cycE. Furthermore, RP-6306 exerted tumor suppressive effects in LMW-E transgenic murine mammary tumors and LMW-E-high TNBC patient-derived xenografts but not in the LMW-E null models examined in parallel. Lastly, transcriptomic and immune profiling demonstrated that RP-6306 treatment induced interferon responses and T-cell infiltration in the LMW-E-high tumor microenvironment, enhancing the antitumor immune response. These findings highlight the LMW-E/PKMYT1/CDK1 regulatory axis as a promising therapeutic target in TNBC, providing the rationale for further clinical development of PKMYT1 inhibitors in this aggressive breast cancer subtype.
ABSTRACT
Cyclin-dependent kinases 4/6 inhibitor (CDK4/6i) plus endocrine therapy (ET) is standard of care for patients with hormone receptor (HR)-positive, HER2-negative metastatic breast cancer (MBC). However, resistance to CDK4/6is plus ET remains a clinical problem with limited therapeutic options following disease progression. Different CDK4/6is might have distinct mechanisms of resistance, and therefore using them sequentially or targeting their differentially altered pathways could delay disease progression. To understand pathways leading to resistance to the CDK4/6is palbociclib and abemaciclib, we generated multiple in vitro models of palbociclib-resistant (PR) and abemaciclib-resistant (AR) cell lines as well as in vivo patient-derived xenografts (PDX) and ex vivo PDX-derived organoids (PDxO) from patients who progressed on CDK4/6i. PR and AR breast cancer cells exhibited distinct transcriptomic and proteomic profiles that sensitized them to different classes of inhibitors; PR cells upregulated G2-M pathways and responded to abemaciclib, while AR cells upregulated mediators of the oxidative phosphorylation pathway (OXPHOS) and responded to OXPHOS inhibitors. PDX and organoid models derived from patients with PR breast cancer remained responsive to abemaciclib. Resistance to palbociclib while maintaining sensitivity to abemaciclib was associated with pathway-specific transcriptional activity but was not associated with any individual genetic alterations. Finally, data from a cohort of 52 patients indicated that patients with HR-positive/HER2-negative MBC who progressed on palbociclib-containing regimens can exhibit a meaningful overall clinical benefit from abemaciclib-based therapy when administered after palbociclib. These findings provide the rationale for clinical trials evaluating the benefit of abemaciclib treatment following progression on a prior CDK4/6i. SIGNIFICANCE: Palbociclib-resistant breast cancers respond to abemaciclib and express pathway-specific signatures of sensitivity, providing a biomarker-driven therapeutic option for patients with metastatic breast cancer following disease progression on cyclin-dependent kinases 4/6 inhibitors.
Subject(s)
Breast Neoplasms , Animals , Humans , Female , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Proteomics , Disease Models, Animal , Disease Progression , Cyclins , Cyclin-Dependent Kinase 4 , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Cyclin-Dependent Kinase 6 , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic useABSTRACT
Treatment strategies with a strong scientific rationale based on specific biomarkers are needed to improve outcomes in patients with advanced sarcomas. Suppression of cell-cycle progression through reactivation of the tumor suppressor retinoblastoma (Rb) using CDK4/6 inhibitors is a potential avenue for novel targeted therapies in sarcomas that harbor intact Rb signaling. Here, we evaluated combination treatment strategies (sequential and concomitant) with the CDK4/6 inhibitor abemacicib to identify optimal combination strategies. Expression of Rb was examined in 1,043 sarcoma tumor specimens, and 50% were found to be Rb-positive. Using in vitro and in vivo models, an effective two-step sequential combination strategy was developed. Abemaciclib was used first to prime Rb-positive sarcoma cells to reversibly arrest in G1 phase. Upon drug removal, cells synchronously traversed to S phase, where a second treatment with S-phase targeted agents (gemcitabine or Wee1 kinase inhibitor) mediated a synergistic response by inducing DNA damage. The response to treatment could be noninvasively monitored using real-time positron emission tomography imaging and serum thymidine kinase activity. Collectively, these results show that a novel, sequential treatment strategy with a CDK4/6 inhibitor followed by a DNA-damaging agent was effective, resulting in synergistic tumor cell killing. This approach can be readily translated into a clinical trial with noninvasive functional imaging and serum biomarkers as indicators of response and cell cycling. SIGNIFICANCE: An innovative sequential therapeutic strategy targeting Rb, followed by treatment with agents that perturb DNA synthesis pathways, results in synergistic killing of Rb-positive sarcomas that can be noninvasively monitored.
Subject(s)
Antineoplastic Agents , Retinal Neoplasms , Retinoblastoma , Sarcoma , Humans , Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinase 6 , DNA , Retinoblastoma/drug therapy , Retinoblastoma Protein/genetics , Sarcoma/metabolismABSTRACT
The identification of biomarker-driven targeted therapies for patients with triple negative breast cancer (TNBC) remains a major clinical challenge, due to a lack of specific targets. Here, we show that cyclin E, a major regulator of G1 to S transition, is deregulated in TNBC and is associated with mutations in DNA repair genes (e.g., BRCA1/2). Breast cancers with high levels of cyclin E not only have a higher prevalence of BRCA1/2 mutations, but also are associated with the worst outcomes. Using several in vitro and in vivo model systems, we show that TNBCs that harbor either mutations in BRCA1/2 or overexpression of cyclin E are very sensitive to the growth inhibitory effects of AZD-1775 (Wee 1 kinase inhibitor) when used in combination with MK-4837 (PARP inhibitor). Combination treatment of TNBC cell lines with these two agents results in synergistic cell killing due to induction of replicative stress, downregulation of DNA repair and cytokinesis failure that results in increased apoptosis. These findings highlight the potential clinical application of using cyclin E and BRCA mutations as biomarkers to select only those patients with the highest replicative stress properties that may benefit from combination treatment with Wee 1 kinase and PARP inhibitors.
ABSTRACT
PURPOSE: Poor prognosis in triple-negative breast cancer (TNBC) is due to an aggressive phenotype and lack of biomarker-driven targeted therapies. Overexpression of cyclin E and phosphorylated-CDK2 are correlated with poor survival in patients with TNBC, and the absence of CDK2 desensitizes cells to inhibition of Wee1 kinase, a key cell-cycle regulator. We hypothesize that cyclin E expression can predict response to therapies, which include the Wee1 kinase inhibitor, AZD1775. EXPERIMENTAL DESIGN: Mono- and combination therapies with AZD1775 were evaluated in TNBC cell lines and multiple patient-derived xenograft (PDX) models with different cyclin E expression profiles. The mechanism(s) of cyclin E-mediated replicative stress were investigated following cyclin E induction or CRISPR/Cas9 knockout by a number of assays in multiple cell lines. RESULTS: Cyclin E overexpression (i) is enriched in TNBCs with high recurrence rates, (ii) sensitizes TNBC cell lines and PDX models to AZD1775, (iii) leads to CDK2-dependent activation of DNA replication stress pathways, and (iv) increases Wee1 kinase activity. Moreover, treatment of cells with either CDK2 inhibitors or carboplatin leads to transient transcriptional induction of cyclin E (in cyclin E-low tumors) and result in DNA replicative stress. Such drug-mediated cyclin E induction in TNBC cells and PDX models sensitizes them to AZD1775 in a sequential treatment combination strategy.Conclusions: Cyclin E is a potential biomarker of response (i) for AZD1775 as monotherapy in cyclin E-high TNBC tumors and (ii) for sequential combination therapy with CDK2 inhibitor or carboplatin followed by AZD1775 in cyclin E-low TNBC tumors.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Cyclin E/genetics , Drug Resistance, Neoplasm/genetics , Gene Expression , Nuclear Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Triple Negative Breast Neoplasms/genetics , Animals , Apoptosis , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Line, Tumor , Cyclic N-Oxides , DNA Repair , DNA Replication , Disease Models, Animal , Humans , Indolizines , Mice , Mice, Knockout , Models, Biological , Prognosis , Pyrazoles/pharmacology , Pyridinium Compounds/pharmacology , Pyrimidinones/pharmacology , Stress, Physiological , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/mortality , Triple Negative Breast Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
Epithelial to mesenchymal transition (EMT) is associated with a wide range of changes in cancer cells, including stemness, chemo- and radio-resistance, and metastasis. The mechanistic role of upstream mediators of EMT has not yet been well characterized. Recently, we showed that non-small cell lung cancers (NSCLC) that have undergone EMT overexpress AXL, a receptor tyrosine kinase. AXL is also overexpressed in a subset of triple-negative breast cancers (TNBC) and head and neck squamous cell carcinomas (HNSCC), and its overexpression has been associated with more aggressive tumor behavior and linked to resistance to chemotherapy, radiotherapy, and targeted therapy. Because the DNA repair pathway is also altered in patient tumor specimens overexpressing AXL, it is hypothesized that modulation of AXL in cells that have undergone EMT will sensitize them to agents targeting the DNA repair pathway. Downregulation or inhibition of AXL directly reversed the EMT phenotype, led to decreased expression of DNA repair genes, and diminished efficiency of homologous recombination (HR) and RAD51 foci formation. As a result, AXL inhibition caused a state of HR deficiency in the cells, making them sensitive to inhibition of the DNA repair protein, PARP1. AXL inhibition synergized with PARP inhibition, leading to apoptotic cell death. AXL expression also associated positively with markers of DNA repair across TNBC, HNSCC, and NSCLC patient cohorts. IMPLICATIONS: The novel role for AXL in DNA repair, linking it to EMT, suggests that AXL can be an effective therapeutic target in combination with targeted therapy such as PARP inhibitors in several different malignancies. Mol Cancer Res; 15(1); 45-58. ©2016 AACR.
Subject(s)
DNA Damage , Neoplasms/enzymology , Neoplasms/pathology , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Proto-Oncogene Proteins/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Apoptosis/drug effects , Cell Line, Tumor , DNA Repair/drug effects , Epithelial-Mesenchymal Transition/drug effects , Humans , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Axl Receptor Tyrosine KinaseABSTRACT
The ubiquitin proteasome-proteolytic pathway has emerged as one of the most significant pathways in modulating protein homeostasis under both normal and disease states. The use of proteasome inhibitors (PI) has played a pivotal role in understanding protein turn over. The main objective of this work was to develop a comprehensive, fast, and reliable, yet simple in vitro assay that would allow for the identification and characterization of a wide range of PIs. The assays consist of a 96-well plate high throughput (HTP) method to assess proteasome activity in Hs578T breast cancer cell extracts, purified 20S proteasome, using a fluorogenic substrate, Suc-leu-leu-val-tyr-7-AMC, specific to the chymotrypsin-like enzymatic activity of the proteasome. We showed that the chymotrypsin-like activity of the proteasome was inhibited in the two in vitro systems, albeit to different degrees. The assay system also includes two cell-based assays consisting of a vector expressing a fusion protein of green fluorescent protein (gfp) and Mouse Ornithine Decarboxylase (MODC) in Zs578T (parental Hs578T carrying the vector that expresses the fusion protein). In the cell-based assay analyses (qualitatively by microscopy and quantitatively by flow cytometry), treatment of Zs578T with PIs prevented the degradation of MODC, accumulated gfp, indicative of increased proteasome inhibition. Because no single assay represents a definitive proof of proteasome inhibitory activity, combined, these assays should serve as a comprehensive benchmark for the identification and partial characterization of novel inhibitors. In summary, the four-step assay protocol can easily be adapted into a high throughput format to rapidly screen unknown inhibitors.