Melissa Dumble

Antitumor Activity of GSK1904529A, a Small-molecule Inhibitor of the Insulin-like Growth Factor-I Receptor Tyrosine Kinase

Purpose: Dysregulation of the insulin-like growth factor-I receptor (IGF-IR) signaling pathway has been implicated in the development of many types of tumors, including prostate, colon, breast, pancreatic, ovarian, and sarcomas. Agents that inhibit IGF-IR activity may be useful in treatment of patients with various cancers. Experimental Design: Kinase assays were used to identify a selective small-molecule inhibitor of IGF-IR activity. The effects of this compound on IGF-IR signaling, cell proliferation, and the cell cycle were determined using a panel of cell lines. Antitumor activity was evaluated in human tumor xenografts growing in athymic mice. Inhibition of IGF-IR and the closely related insulin receptor (IR) was measured in vivo, and the effect on glucose metabolism was evaluated. Results: GSK1904529A selectively inhibits IGF-IR and IR with IC50s of 27 and 25 nmol/L, respectively. GSK1904529A blocks receptor autophosphorylation and downstream signaling, leading to cell cycle arrest. It inhibits the proliferation of cell lines derived from solid and hematologic malignancies, with multiple myeloma and Ewings sarcoma cell lines being most sensitive. Oral administration of GSK1904529A decreases the growth of human tumor xenografts in mice, consistent with a reduction of IGF-IR phosphorylation in tumors. Despite the potent inhibitory activity of GSK1904529A on IR in vitro and in vivo, minimal effects on blood glucose levels are observed in animals at doses that show significant antitumor activity. Conclusion: GSK1904529A is a promising candidate for therapeutic use in IGF-IR–dependent tumors.

Bridging the Gap between Cytotoxic and Biologic Therapy with Metronomic Topotecan and Pazopanib in Ovarian Cancer

This study aimed to investigate the antitumor and antiangiogenic effects utilizing a novel therapy regimen of metronomic topotecan and pazopanib, a multireceptor tyrosine kinase inhibitor. In vitro (Western blot) and in vivo dose-finding experiments were done following pazopanib therapy in ovarian cancer models. Pazopanib and metronomic (daily) oral topotecan therapy was examined in an orthotopic model of ovarian cancer. Tumor weights, survival, and markers of the tumor microenvironment [angiogenesis (CD31 and pericyte coverage), proliferation (Ki-67), and apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling)] were analyzed by immunostaining following therapy. Pazopanib therapy reduced vascular endothelial growth factor receptor 2 (VEGFR-2) activity in vitro and vivo in a dose-dependent manner. Compared with control mice, pazopanib reduced tumor weight by 28% to 82% (P < 0.01 in the SKOV3ip1 model) and metronomic topotecan reduced tumor weight by 40% to 59% in the HeyA8 (P = 0.13) and SKOV3ip1 (P = 0.07) models. Combination therapy had the greatest effect with 79% to 84% reduction (P < 0.01 for both models). In the SKOV3ip1 and A2780 models, mouse survival was significantly longer (P < 0.001 versus controls) with pazopanib and metronomic topotecan therapy. Pazopanib therapy reduced murine endothelial cell migration in vitro in a dose-dependent manner following VEGF stimulation and decreased tumor microvessel density and pericyte coverage when given in combination with metronomic topotecan. Tumor cell proliferation decreased in all treatment arms compared with controls (P < 0.01 for combination groups) and increased tumor cell apoptosis by 4-fold with combination therapy. Pazopanib therapy in combination with metronomic topotecan therapy showed significant antitumor and antiangiogenic properties in preclinical ovarian cancer models and warrants further investigation as a novel therapeutic regimen in clinical trials. Mol Cancer Ther; 9(4); 985–95. ©2010 AACR.

Discovery of Novel AKT Inhibitors with Enhanced Anti-Tumor Effects in Combination with the MEK Inhibitor

Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via PI3K/PTEN mutation or loss) while cell lines with activating mutations in the MAPK pathway (KRAS/BRAF) were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib) resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.

Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors.

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

Rpl27a mutation in the sooty foot ataxia mouse phenocopies high p53 mouse models.

Ribosomal stress is an important, yet poorly understood, mechanism that results in activation of the p53 tumour suppressor. We present a mutation in the ribosomal protein Rpl27a gene (sooty foot ataxia mice), isolated through a sensitized N‐ethyl‐N‐nitrosourea (ENU) mutagenesis screen for p53 pathway defects, that shares striking phenotypic similarities with high p53 mouse models, including cerebellar ataxia, pancytopenia and epidermal hyperpigmentation. This phenocopy is rescued in a haploinsufficient p53 background. A detailed examination of the bone marrow in these mice identified reduced numbers of haematopoietic stem cells and a p53‐dependent c‐Kit down‐regulation. These studies suggest that reduced Rpl27a increases p53 activity in vivo, further evident with a delay in tumorigenesis in mutant mice. Taken together, these data demonstrate that Rpl27a plays a crucial role in multiple tissues and that disruption of this ribosomal protein affects both development and transformation. Copyright

Abstract 5517: PTC596-induced Bmi1 hyper-phosphorylation via Cdk1/2 activation resulting in tumor stem cell depletion

The Polycomb group (PcG) transcription repressor BMI1 is highly expressed in human cancers and is required for the clonogenic self-renewal and tumorigenesis of human cancer cells including those in hematological cancer and neuroblastoma. PTC596 is efficacious in vivo across a range of xenograft tumor models, including models of glioblastoma, fibrosarcoma and leukemia as well as orthotopic models of GBM. With EC50 values of 30-200 nM in a variety of tumor cell lines, PTC596 selectively reduces the level of functional BMI1 protein resulting in the depletion of the tumor stem cell fraction. PTC596 induces the hyper-phosphorylation of BMI1 leading to its degradation and the reduction of polycomb repressive complex 1 (PRC1) activity. Mechanistic studies suggest that PTC596 inhibits APC/CCDC20 activity resulting in the persistent activation of CDK1 and CDK2 which mediate the hyperphosphorylation of BMI1. Studies are ongoing to elucidate the mechanism of PTC596 inhibition of APC/CCDC20 and its preferential depletion of the tumor stem cell fraction. Citation Format: Min Jung Kim, Liangxian Cao, Josephine Sheedy, Nicole Risher, Melissa Dumble, Chang-Sun Lee, Nadiya Sydorenko, Ramil Baiazitov, Wu Du, Young-Choon Moon, Marla L. Weetall, Joseph Colacino, Thomas W. Davis. PTC596-induced Bmi1 hyper-phosphorylation via Cdk1/2 activation resulting in tumor stem cell depletion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5517. doi:10.1158/1538-7445.AM2014-5517

Evaluating the mechanism and therapeutic potential of PTC-028, a novel inhibitor of BMI-1 function in ovarian cancer

BMI-1, also known as a stem cell factor, is frequently upregulated in several malignancies. Elevated expression of BMI-1 correlates with poor prognosis and is therefore considered a viable therapeutic target in a number of malignancies including ovarian cancer. Realizing the immense pathologic significance of BMI-1, small-molecule inhibitors against BMI-1 are recently being developed. In this study, we functionally characterize PTC-028, an orally bioavailable compound that decreases BMI-1 levels by posttranslational modification. We report that PTC-028 treatment selectively inhibits cancer cells in clonal growth and viability assays, whereas normal cells remain unaffected. Mechanistically, hyperphosphorylation-mediated depletion of cellular BMI-1 by PTC-028 coupled with a concurrent temporal decrease in ATP and a compromised mitochondrial redox balance potentiates caspase-dependent apoptosis. In vivo, orally administered PTC-028, as a single agent, exhibits significant antitumor activity comparable with the standard cisplatin/paclitaxel therapy in an orthotopic mouse model of ovarian cancer. Thus, PTC-028 has the potential to be used as an effective therapeutic agent in patients with epithelial ovarian cancer, where treatment options are limited. Mol Cancer Ther; 17(1); 39–49. ©2017 AACR.

Discovery and Optimization of Indolyl-Containing 4-Hydroxy-2-Pyridone Type II DNA Topoisomerase Inhibitors Active against Multidrug Resistant Gram-negative Bacteria

There exists an urgent medical need to identify new chemical entities (NCEs) targeting multidrug resistant (MDR) bacterial infections, particularly those caused by Gram-negative pathogens. 4-Hydroxy-2-pyridones represent a novel class of nonfluoroquinolone inhibitors of bacterial type II topoisomerases active against MDR Gram-negative bacteria. Herein, we report on the discovery and structure–activity relationships of a series of fused indolyl-containing 4-hydroxy-2-pyridones with improved in vitro antibacterial activity against fluoroquinolone resistant strains. Compounds 6o and 6v are representative of this class, targeting both bacterial DNA gyrase and topoisomerase IV (Topo IV). In an abbreviated susceptibility screen, compounds 6o and 6v showed improved MIC90 values against Escherichia coli (0.5–1 μg/mL) and Acinetobacter baumannii (8–16 μg/mL) compared to the precursor compounds. In a murine septicemia model, both compounds showed complete protection in mice infected with a lethal dose of E. coli.

Abstract A22: Preclinical evaluation of Bmi1 inhibition in pancreatic ductal adenocarcinoma

Pancreatic Ductal Adenocarcinoma (PDA) is the fourth leading cause of cancer-related deaths in the United States with a five-year survival rate of 6% and a rising mortality rate. The majority of patients present with advanced disease that is unresponsive to chemotherapy, highlighting the need for novel therapeutic strategies. Bmi1, a member of the Polycomb Group (PcG) family of transcriptional repressors, is known to be upregulated in PDA beginning in early pre-malignancy. Elevated Bmi1 expression in pancreatic tumors has been shown to correlate with poor prognosis, increased metastasis, and chemotherapy resistance in patients. Additionally, a recent study found Bmi1 to be required for pancreatic neoplasia in a murine model of PDA. Together, these data support the hypothesis that Bmi1 promotes the growth and survival of pancreatic tumors, suggesting it may be a novel therapeutic target. Bmi1 is a critical member of the PcG PRC1 complex, where it interacts with E3 ligase Ring1B in a dose-dependent manner to promote mono-ubiquitination of H2AK119, a histone modification that results in repression of gene expression. Among the best-characterized functions of Bmi1 is its negative regulation of the cdkn2a locus, which encodes two tumor suppressor proteins and cell cycle regulators, p16Ink4a and p14Arf. This is notable, as p16Ink4a is inactivated in most pancreatic tumors, often through epigenetic mechanisms. More recently, Bmi1 was found to have an additional role in G2/M checkpoint regulation following double strand breaks (DSBs). Knockdown of Bmi1 in breast and prostate cancer cell lines treated with etoposide to induce DSBs was found to induce G2/M arrest, while Bmi1 overexpression reduced G2/M arrest. This effect was mediated by reduced ATM activation. Based on these data, we decided to evaluate the effect of inhibiting Bmi1 both in cultured pancreatic tumor cells and in vivo using a genetically engineered mouse model of pancreatic ductal adenocarcinoma. PTC596 is an orally available, small-molecule inhibitor of Bmi1 developed by PTC Therapeutics that inhibits proliferation of human and murine PDA cell lines in vitro. Cell cycle analysis of PTC596-treated PDA cells supports G2/M arrest as a mechanism of inhibition. PTC596 is also well tolerated in vivo and can successfully be delivered to tumor tissues of treated mice. We are currently carrying out an intervention study in the KPC (K-rasLSL.G12D/+; p53LSL.R172H/+; Pdx1-Cre) genetically engineered mouse model of PDA to evaluate whether treatment with PTC596, alone or in combination with gemcitabine, confers a survival advantage or alters tumor growth kinetics. Preliminary results support slowed tumor growth and elevated levels of apoptosis in tumor tissues of treated mice. Ongoing work focuses on identifying the mechanism(s) of the observed effects on pancreatic tumors using tissues from mice treated with PTC596 as well as PDA cells with inducible knockdown of Bmi1. Citation Format: Jaime A. Eberle, Haoxuan Yuan, Carmine Palermo, Stephen A. Sastra, Liangxian Cao, Young-choon Moon, Melissa Dumble, Thomas Davis, Kenneth P. Olive. Preclinical evaluation of Bmi1 inhibition in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A22.

Abstract A130: Targeting BMI-1 by the novel BMI-1 inhibitor PTC596 in acute leukemia

Leukemia stem cells are resistant to chemotherapeutic agents and contribute to disease relapse. BMI-1, a part of polycomb repressive complex 1 (PRC1) is essential for the self-renewal of normal hematopoietic and leukemia stem cells. We have reported that acute myeloid leukemia (AML) patients with higher levels of BMI-1 have worse overall survival and that the translational BMI-1 inhibitor PTC-209 induces apoptosis in patient-derived CD34+CD38low/- AML cells. PTC596 is a second-generation BMI-1 inhibitor that accelerates BMI-1 degradation. Based on preclinical data showing excellent anti-tumor activities against solid cancers, PTC596 is currently in PH1 clinical development. We here investigated preclinical activities of PTC596 against acute leukemias in vitro and in vivo. A total of 6 AML (MOLM-13, OCI-AML3, U-937, MV4-11, MOLM-14 and HL60) and 3 acute lymphoblastic leukemia (ALL) (Reh, NALM6 and MOLT-4) cell lines were exposed to PTC596 for 48 hours. PTC596 exhibited dose- and time-dependent anti-proliferative and cytotoxic (apoptotic) activities. The IC50 values (concentration at which cell growth is inhibited by 50% at 48 hours of exposure) were 30.7 ± 4.1 nM (mean ± SEM) for AML and 37.7 ± 8.0 nM for ALL. The ED50 values (effective concentration inducing 50% killing as measured by Annexin V/PI induction) were 60.3 ± 6.7 nM for AML and 77.8 ± 11.5 nM for ALL. PTC596 induced apoptosis in AML cells irrespective of their p53 status. 72-hour treatment of MOLM-13 and OCI-AML3 cells with 100nM PTC596 reduced total BMI-1 protein levels by 87% and 61%, respectively. Forced overexpression of BMI-1 in K562 cells abrogated PTC596-induced apoptosis (∼ 80% at 50 nM PTC596; P Citation Format: Yuki Nishida, Aya Maeda, Liangxian Cao, Melissa Dumble, Shinya Kimura, Thomas W. Davis, Kensuke Kojima. Targeting BMI-1 by the novel BMI-1 inhibitor PTC596 in acute leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A130.