Richard P. Beckmann

Efficacy and Safety of Abemaciclib, an Inhibitor of CDK4 and CDK6, for Patients with Breast Cancer, Non-Small Cell Lung Cancer, and Other Solid Tumors.

UNLABELLED We evaluated the safety, pharmacokinetic profile, pharmacodynamic effects, and antitumor activity of abemaciclib, an orally bioavailable inhibitor of cyclin-dependent kinases (CDK) 4 and 6, in a multicenter study including phase I dose escalation followed by tumor-specific cohorts for breast cancer, non-small cell lung cancer (NSCLC), glioblastoma, melanoma, and colorectal cancer. A total of 225 patients were enrolled: 33 in dose escalation and 192 in tumor-specific cohorts. Dose-limiting toxicity was grade 3 fatigue. The maximum tolerated dose was 200 mg every 12 hours. The most common possibly related treatment-emergent adverse events involved fatigue and the gastrointestinal, renal, or hematopoietic systems. Plasma concentrations increased with dose, and pharmacodynamic effects were observed in proliferating keratinocytes and tumors. Radiographic responses were achieved in previously treated patients with breast cancer, NSCLC, and melanoma. For hormone receptor-positive breast cancer, the overall response rate was 31%; moreover, 61% of patients achieved either response or stable disease lasting ≥6 months. SIGNIFICANCE Abemaciclib represents the first selective inhibitor of CDK4 and CDK6 with a safety profile allowing continuous dosing to achieve sustained target inhibition. This first-in-human experience demonstrates single-agent activity for patients with advanced breast cancer, NSCLC, and other solid tumors. Cancer Discov; 6(7); 740-53. ©2016 AACR.See related commentary by Lim et al., p. 697This article is highlighted in the In This Issue feature, p. 681.

Mitotic requirement for aurora A kinase is bypassed in the absence of aurora B kinase

We investigated why treatment of cells with dual aurora A and B kinase inhibitors produces phenotypes identical to inactivation of aurora B. We found that dual aurora kinase inhibitors in fact potently inhibit cellular activities of both kinases, indicating that inactivation of aurora B bypasses aurora A in mitosis. RNAi experiments further established that inactivation of aurora B indeed bypasses the requirement for aurora A and leads to polyploidy. Inactivation of aurora A activates checkpoint kinase BubR1 in an aurora B‐dependent manner. Our results thus show that aurora B is responsible for mitotic arrest in the absence of aurora A.

CHEK again: revisiting the development of CHK1 inhibitors for cancer therapy.

CHEK1 encodes the serine/threonine kinase CHK1, a central component of the DNA damage response. CHK1 regulates cell cycle checkpoints following genotoxic stress to prevent the entry of cells with damaged DNA into mitosis and coordinates various aspects of DNA repair. Accordingly, CHK1 has become a target of considerable interest in oncology. CHK1 inhibitors potentiate the efficacy of DNA-damaging chemotherapeutics by abrogating CHK1-mediated cell cycle arrest and preventing repair of damaged DNA. In addition, CHK1 inhibitors interfere with the biological role of CHK1 as a principal regulator of the cell cycle that controls the initiation of DNA replication, stabilizes replication forks, and coordinates mitosis. Since these functions of CHK1 facilitate progression through an unperturbed cell cycle, CHK1 inhibitors are being developed not only as chemopotentiators, but also as single-agent therapies. This review is intended to provide information on the current progress of CHK1 inhibitors in pre-clinical and clinical development and will focus on mechanisms of single-agent activity and potential strategies for patient tailoring and combinations with non-genotoxic agents.

SUBCELLULAR LOCALIZATION AND ANALYSIS OF APPARENT 180-KDA AND 220-KDA PROTEINS OF THE BREAST CANCER SUSCEPTIBILITY GENE, BRCA1

The breast cancer susceptibility gene BRCA1 encodes an 1863-amino acid protein that acts as a tumor suppressor. The biochemical function of BRCA1 is unknown, and there are conflicting results describing its subcellular location. We have identified a 220-kDa protein, which is reactive with three antibodies raised against the amino- and carboxyl-terminal regions of BRCA1. Immunoflourescence staining with an antibody to the carboxyl terminus of BRCA1 localized the protein to the nucleus of breast, ovarian, and cervical carcinoma-derived cell lines. A similar result was observed by biochemical subcellular fractionation that indicated that the 220-kDa protein was localized primarily to the nucleus of cell lines established from breast carcinomas. In addition to the 220-kDa protein, one antibody, C-20, also recognized a 180-kDa protein in MDA-MB-468 total cell lysates that was not detected by the other two antibodies. Several observations suggest the 180-kDa protein is the epidermal growth factor (EGF) receptor: (i) C-20 reacted avidly with a 180-kDa protein immunoprecipitated by an antibody to the EGF receptor; (ii) an EGF receptor antibody detected a 180-kDa protein immunoprecipitated by C-20; (iii) the affinity purified EGF receptor was both immunoprecipitated and detected on immunoblots by the C-20 antibody but not another BRCA1 antibody; (iv) similar phosphopeptide maps were generated from the EGF receptor and the 180-kDa protein immunoprecipitated by C-20, and this peptide map was distinct from the 220-kDa phosphoprotein; and (v) the C-20 immunizing peptide bears sequence identity to the EGF receptor. These results indicate that BRCA1 is a 220-kDa nuclear protein and that the 180-kDa protein reported previously may be unrelated to BRCA1.

Semi-Mechanistic Pharmacokinetic/Pharmacodynamic Modeling of the Antitumor Activity of LY2835219, a New Cyclin-Dependent Kinase 4/6 Inhibitor, in Mice Bearing Human Tumor Xenografts

Purpose: Selective inhibition of cyclin-dependent kinases 4 and 6 (CDK4/6) represents a promising therapeutic strategy. However, despite documented evidence of clinical activity, limited information is available on the optimal dosing strategy of CDK4/6 inhibitors. Here, we present an integrated semi-mechanistic pharmacokinetic/pharmacodynamic model to characterize the quantitative pharmacology of LY2835219, a CDK4/6 inhibitor, in xenograft tumors. Experimental Design: LY2835219 plasma concentrations were connected to CDK4/6 inhibition and cell-cycle arrest in colo-205 human colorectal xenografts by incorporating the biomarkers, phospho-(ser780)-Rb, topoisomerase II α, and phosphohistone H3, into a precursor-dependent transit compartment model. This biomarker model was then connected to tumor growth inhibition (TGI) by: (i) relating the rate of tumor growth to mitotic cell density, and (ii) incorporating a concentration-dependent mixed cytostatic/cytotoxic effect driving quiescence and cell death at high doses. Model validation was evaluated by predicting LY2835219-mediated antitumor effect in A375 human melanoma xenografts. Results: The model successfully described LY2835219-mediated CDK4/6 inhibition, cell-cycle arrest, and TGI in colo-205, and was validated in A375. The model also demonstrated that a chronic dosing strategy achieving minimum steady-state trough plasma concentrations of 200 ng/mL is required to maintain durable cell-cycle arrest. Quiescence and cell death can be induced by further increasing LY2835219 plasma concentrations. Conclusions: Our model provides mechanistic insight into the quantitative pharmacology of LY2835219 and supports the therapeutic dose and chronic dosing strategy currently adopted in clinical studies. Clin Cancer Res; 20(14); 3763–74. ©2014 AACR.

The CDK4/6 Inhibitor LY2835219 Overcomes Vemurafenib Resistance Resulting from MAPK Reactivation and Cyclin D1 Upregulation

B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanoma patients with B-RAF V600E mutation. However, most patients treated with vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently, we found that MAPK is reactivated and cyclin D1 is elevated in vemurafenib-resistant tumors, as well as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1–CDK4/6 signaling by LY2835219 is an effective strategy to overcome MAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma. Mol Cancer Ther; 13(10); 2253–63. ©2014 AACR.

Short pigment epithelial-derived factor-derived peptide inhibits angiogenesis and tumor growth.

Purpose: Pigment epithelial-derived factor (PEDF) is a potent angiogenesis inhibitor with multiple other functions, some of which enhance tumor growth. Our previous studies mapped PEDF antiangiogenic and prosurvival activities to distinct epitopes. This study was aimed to determine the minimal fragment of PEDF, which maintains antiangiogenic and antitumor efficacy. Experimental Design: We analyzed antigenicity, hydrophilicity, and charge distribution of the angioinhibitory epitope (the 34-mer) and designed three peptides covering its COOH terminus, P14, P18, and P23. We analyzed their ability to block endothelial cell chemotaxis and induce apoptosis in vitro and their antiangiogenic activity in vivo. The selected peptide was tested for the antitumor activity against mildly aggressive xenografted prostate carcinoma and highly aggressive renal cell carcinoma. To verify that P18 acts in the same manner as PEDF, we used immunohistochemistry to measure PEDF targets, vascular endothelial growth factor receptor 2, and CD95 ligand expression in P18-treated vasculature. Results: P14 and P18 blocked endothelial cell chemotaxis; P18 and P23 induced apoptosis. P18 showed the highest IC50 and blocked angiogenesis in vivo: P23 was inactive and P14 was proangiogenic. P18 increased the production of CD95 ligand and reduced the expression of vascular endothelial growth factor receptor 2 by the endothelial cells in vivo. In tumor studies, P18 was more effective in blocking the angiogenesis and growth of the prostate cancer than parental 34-mer; in the renal cell carcinoma, P18 strongly decreased angiogenesis and halted the progression of established tumors. Conclusions: P18 is a novel and potent antiangiogenic biotherapeutic agent that has potential to be developed for the treatment of prostate and renal cancer.

Cdk1 Activity Is Required for Mitotic Activation of Aurora A during G2/M Transition of Human Cells

In mammalian cells entry into and progression through mitosis are regulated by multiple mitotic kinases. How mitotic kinases interact with each other and coordinately regulate mitosis remains to be fully understood. Here we employed a chemical biology approach using selective small molecule kinase inhibitors to dissect the relationship between Cdk1 and Aurora A kinases during G2/M transition. We find that activation of Aurora A first occurs at centrosomes at late G2 and is required for centrosome separation independently of Cdk1 activity. Upon entry into mitosis, Aurora A then becomes fully activated downstream of Cdk1 activation. Inactivation of Aurora A or Plk1 individually during a synchronized cell cycle shows no significant effect on Cdk1 activation and entry into mitosis. However, simultaneous inactivation of both Aurora A and Plk1 markedly delays Cdk1 activation and entry into mitosis, suggesting that Aurora A and Plk1 have redundant functions in the feedback activation of Cdk1. Together, our data suggest that Cdk1, Aurora A, and Plk1 mitotic kinases participate in a feedback activation loop and that activation of Cdk1 initiates the feedback loop activity, leading to rapid and timely entry into mitosis in human cells. In addition, live cell imaging reveals that the nuclear cycle of cells becomes uncoupled from cytokinesis upon inactivation of both Aurora A and Aurora B kinases and continues to oscillate in a Cdk1-dependent manner in the absence of cytokinesis, resulting in multinucleated, polyploidy cells.

LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms

CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1s role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe. Mol Cancer Ther; 14(9); 2004–13. ©2015 AACR.

Conditional transformation of rat embryo fibroblast cells by a cyclin D1-cdk4 fusion gene.

Cyclin D1 gene overexpression is a frequent event in a number of human cancers. These observations have led to the suggestion that cyclin D1 alterations might play a role in the etiology of cancer. This possibility is supported by the finding that transfection of mammalian cells with cyclin D1 can accelerate progression through the G1 phase of the cell cycle. Moreover, cyclin D1 can function as an oncogene by cooperating with activated Ha-ras to transform primary rat embryo fibroblasts (REFs). In addition, cyclin D1 transgenics develop hyperplasia and neoplasia of the thymus and mammary gland. We have constructed a novel fusion gene consisting of full-length human cyclin D1 and cdk4 genes. This fusion gene was expressed in insect cells and the fusion protein was shown to be enzymatically active. The fusion gene was expressed in mammalian cells under the control of tet-repressor. This fusion gene immortalized primary REFs, and cooperated with activated Ha-ras to transform primary REFs, in terms of anchorage-independent growth in vitro and formation of tumors in vivo. Utilizing a tet-regulated gene expression system, we have shown that proliferation of stably transfected primary REFs in vitro and in vivo is dependent on the continued expression of the cyclin D1-cdk4 fusion gene. These cell lines could be useful in the discovery of novel cancer therapeutics to modulate cyclin D1.cdk4 activity.