Thomas McGonigal

ABT-888, an Orally Active Poly(ADP-Ribose) Polymerase Inhibitor that Potentiates DNA-Damaging Agents in Preclinical Tumor Models

Purpose: To evaluate the preclinical pharmacokinetics and antitumor efficacy of a novel orally bioavailable poly(ADP-ribose) polymerase (PARP) inhibitor, ABT-888. Experimental Design:In vitro potency was determined in a PARP-1 and PARP-2 enzyme assay. In vivo efficacy was evaluated in syngeneic and xenograft models in combination with temozolomide, platinums, cyclophosphamide, and ionizing radiation. Results: ABT-888 is a potent inhibitor of both PARP-1 and PARP-2 with Kis of 5.2 and 2.9 nmol/L, respectively. The compound has good oral bioavailability and crosses the blood-brain barrier. ABT-888 strongly potentiated temozolomide in the B16F10 s.c. murine melanoma model. PARP inhibition dramatically increased the efficacy of temozolomide at ABT-888 doses as low as 3.1 mg/kg/d and a maximal efficacy achieved at 25 mg/kg/d. In the 9L orthotopic rat glioma model, temozolomide alone exhibited minimal efficacy, whereas ABT-888, when combined with temozolomide, significantly slowed tumor progression. In the MX-1 breast xenograft model (BRCA1 deletion and BRCA2 mutation), ABT-888 potentiated cisplatin, carboplatin, and cyclophosphamide, causing regression of established tumors, whereas with comparable doses of cytotoxic agents alone, only modest tumor inhibition was exhibited. Finally, ABT-888 potentiated radiation (2 Gy/d × 10) in an HCT-116 colon carcinoma model. In each model, ABT-888 did not display single-agent activity. Conclusions: ABT-888 is a potent inhibitor of PARP, has good oral bioavailability, can cross the blood-brain barrier, and potentiates temozolomide, platinums, cyclophosphamide, and radiation in syngeneic and xenograft tumor models. This broad spectrum of chemopotentiation and radiopotentiation makes this compound an attractive candidate for clinical evaluation.

Potent and selective inhibitors of Akt kinases slow the progress of tumors in vivo

The Akt kinases are central nodes in signal transduction pathways that are important for cellular transformation and tumor progression. We report the development of a series of potent and selective indazole-pyridine based Akt inhibitors. These compounds, exemplified by A-443654 (Ki = 160 pmol/L versus Akt1), inhibit Akt-dependent signal transduction in cells and in vivo in a dose-responsive manner. In vivo, the Akt inhibitors slow the progression of tumors when used as monotherapy or in combination with paclitaxel or rapamycin. Tumor growth inhibition was observed during the dosing interval, and the tumors regrew when compound administration was ceased. The therapeutic window for these compounds is narrow. Efficacy is achieved at doses ∼2-fold lower than the maximally tolerated doses. Consistent with data from knockout animals, the Akt inhibitors induce an increase in insulin secretion. They also induce a reactive increase in Akt phosphorylation. Other toxicities observed, including malaise and weight loss, are consistent with abnormalities in glucose metabolism. These data show that direct Akt inhibition may be useful in cancer therapy, but significant metabolic toxicities are likely dose limiting.

Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-beta-glucosyltransferase.

The BGL2 gene encodes a unique 1,3-beta-glucosyltransferase (Bgl2p) present in the cell wall of Candida albicans and other fungi. Although believed to be involved in cell wall assembly, disruption of the gene in saccharomyces cerevisiae showed no apparent phenotype. We performed sequential disruptions of the BGL2 loci in a homozygous ura3 clinical isolate of C. albicans using the URA3 blaster method, in order to investigate the role of Bgl2p in this dimorphic, pathogenic fungus. Strain CACW-1 contained disruptions of both homologues of the BGL2 gene and lacked Bgl2p, as assessed by protein extraction, SDS-PAGE and Western blot analysis, and enzyme assay; however, residual non-Bgl2p transferase activity was detected. CACW-1 was attenuated in virulence for mice when compared to an isogenic parent strain, and fewer organisms were recovered from the kidneys of infected animals. Additional phenotypic changes included: (1) a dramatic increase in the sensitivity to the chitin synthesis inhibitor nikkomycin Z when CACW-1 cells were incubated at 37 or 42 degrees C; (2) an 8.7 +/- 1.6% slower growth rate at 37 degrees C for CACW-1 when compared to its isogenic parent; and (3) aggregation of CACW-1 cells during stationary phase and/or incubation of stationary phase cells in phosphate buffer. Characterization of SDS-extracted cell walls did not reveal any significant differences in the levels of 1,3-beta- or 1,6-beta-glucan. These data reveal that loss of Bgl2p does have a phenotype in C. albicans, and indicate that (1) loss of Bgl2p function renders cells more dependent on chitin for wall integrity, and attenuates virulence (probably due to subtle changes in wall structure), and (2) that additional 1,3-beta-glucosyltransferases are present in the C. albicans BGL2 disruptant.

Acquired Resistance to Combination Treatment with Temozolomide and ABT-888 Is Mediated by Both Base Excision Repair and Homologous Recombination DNA Repair Pathways

Many established cancer therapies involve DNA-damaging chemotherapy or radiotherapy. Gain of DNA repair capacity of the tumor represents a common mechanism used by cancer cells to survive DNA-damaging therapy. Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that is activated by DNA damage and plays a critical role in base excision repair. Inhibition of PARP represents an attractive approach for the treatment of cancer. Previously, we have described the discovery and characterization of a potent PARP inhibitor, ABT-888. ABT-888 potentiates the activity of DNA-damaging agents such as temozolomide (TMZ) in a variety of preclinical models. We report here the generation of HCT116 cells resistant to treatment with TMZ and ABT-888 (HCT116R cells). HCT116R cells exhibit decreased H2AX phosphorylation in response to treatment with TMZ and ABT-888 relative to parental HCT116 cells. Microarray and Western blot studies indicate that HCT116R cells have decreased PARP-1 and elevated Rad51 expression levels. HCT116R cells are dependent on Rad51 for proliferation and survival, as shown by inhibition of proliferation and induction of apoptosis upon treatment with Rad51 small interfering RNA. In addition, HCT116R cells are more resistant to radiation than the parental HCT116 cells. Our study suggests that cancer cells upregulate the homologous recombination DNA repair pathway to compensate for the loss of base excision repair, which may account for the observed resistance to treatment with TMZ and ABT-888. (Mol Cancer Res 2009;7(10):1686–92)

Role of focal adhesion kinase in human cancer: a potential target for drug discovery.

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to the points of cell contact with the extracellular matrix, called focal adhesions. FAK is involved in several cellular processes including invasion, motility, proliferation and apoptosis. In in vivo animal studies, FAK has been shown to contribute to tumor development and malignancy. Furthermore, FAK expression was shown to be elevated in a number of human cancers. Increased FAK expression and activity are correlated with malignant phenotype and poor prognosis in patients. Taken together, these studies suggest that FAK is a potentially good target for drug discovery. In this review, FAK and its relationship to cancer, as well as approaches to therapeutic intervention of FAK will be discussed.

Clearance of systemic hematologic tumors by venetoclax (Abt-199) and navitoclax.

The Bcl‐2 family inhibitors venetoclax and navitoclax demonstrated potent antitumor activity in chronic lymphocytic leukemia patients, notably in reducing marrow load and adenopathy. Subsequent trials with venetoclax have been initiated in non‐Hodgkins lymphoma and multiple myeloma patients. Traditional preclinical models fall short either in faithfully recapitulating disease progression within such compartments or in allowing the direct longitudinal analysis of systemic disease. We show that intravenous inoculation of engineered RS4;11 (acute lymphoblastic leukemia) and Granta 519 (mantle cell lymphoma) bioluminescent reporter cell lines result in tumor engraftment of bone marrow, with additional invasion of the central nervous system in the case of Granta 519. Importantly, apoptosis induction and response of these systemically engrafted tumors to Bcl‐2 family inhibitors alone or in combination with standard‐of‐care agents could be monitored longitudinally with optical imaging, and was more accurately reflective of the observed clinical response.

Construction of a human DNA library in a circular centromere-based yeast plasmid.

The construction of a human DNA library in a centromere-based circular yeast plasmid is described. The vector contains the yeast CEN3 sequence, the URA3 gene for propagation in yeast and a hygromycin-resistance gene (HyR) for selection in mammalian cells. The library consists of 64,000 members with an average insert size of 150 kb, with some members containing inserts of > 1 Mb. We calculate that the library contains three human genome equivalents of DNA. Clones can be identified by a PCR-based screening of DNA pools from individual colonies that have been stored in microtiter wells.

LRRC15 is a novel mesenchymal protein and stromal target for antibody-drug conjugates.

Progress in understanding tumor stromal biology has been constrained in part because cancer-associated fibroblasts (CAF) are a heterogeneous population with limited cell-type-specific protein markers. Using RNA expression profiling, we identified the membrane protein leucine-rich repeat containing 15 (LRRC15) as highly expressed in multiple solid tumor indications with limited normal tissue expression. LRRC15 was expressed on stromal fibroblasts in many solid tumors (e.g., breast, head and neck, lung, pancreatic) as well as directly on a subset of cancer cells of mesenchymal origin (e.g., sarcoma, melanoma, glioblastoma). LRRC15 expression was induced by TGFβ on activated fibroblasts (αSMA+) and on mesenchymal stem cells. These collective findings suggested LRRC15 as a novel CAF and mesenchymal marker with utility as a therapeutic target for the treatment of cancers with LRRC15-positive stromal desmoplasia or cancers of mesenchymal origin. ABBV-085 is a monomethyl auristatin E (MMAE)-containing antibody-drug conjugate (ADC) directed against LRRC15, and it demonstrated robust preclinical efficacy against LRRC15 stromal-positive/cancer-negative, and LRRC15 cancer-positive models as a monotherapy, or in combination with standard-of-care therapies. ABBV-085s unique mechanism of action relied upon the cell-permeable properties of MMAE to preferentially kill cancer cells over LRRC15-positive CAF while also increasing immune infiltrate (e.g., F4/80+ macrophages) in the tumor microenvironment. In summary, these findings validate LRRC15 as a novel therapeutic target in multiple solid tumor indications and support the ongoing clinical development of the LRRC15-targeted ADC ABBV-085.Significance: These findings identify LRRC15 as a new marker of cancer-associated fibroblasts and cancers of mesenchymal origin and provide preclinical evidence for the efficacy of an antibody-drug conjugate targeting the tumor stroma. Cancer Res; 78(14); 4059-72. ©2018 AACR.

The Volume of Three-Dimensional Cultures of Cancer Cells In Vitro Influences Transcriptional Profile Differences and Similarities with Monolayer Cultures and Xenografted Tumors

Improving the congruity of preclinical models with cancer as it is manifested in humans is a potential way to mitigate the high attrition rate of new cancer therapies in the clinic. In this regard, three-dimensional (3D) tumor cultures in vitro have recently regained interest as they have been acclaimed to have higher similarity to tumors in vivo than to cells grown in monolayers (2D). To identify cancer functions that are active in 3D rather than in 2D cultures, we compared the transcriptional profiles (TPs) of two non-small cell lung carcinoma cell lines, NCI-H1650 and EBC-1 grown in both conditions to the TP of xenografted tumors. Because confluence, diameter or volume can hypothetically alter TPs, we made intra- and inter-culture comparisons using samples with defined dimensions. As projected by Ingenuity Pathway Analysis (IPA), a limited number of signal transduction pathways operational in vivo were better represented by 3D than by 2D cultures in vitro. Growth of 2D and 3D cultures as well as xenografts induced major changes in the TPs of these 3 modes of culturing. Alterations of transcriptional network activation that were predicted to evolve similarly during progression of 3D cultures and xenografts involved the following functions: hypoxia, proliferation, cell cycle progression, angiogenesis, cell adhesion, and interleukin activation. Direct comparison of TPs of 3D cultures and xenografts to monolayer cultures yielded up-regulation of networks involved in hypoxia, TGF and Wnt signaling as well as regulation of epithelial mesenchymal transition. Differences in TP of 2D and 3D cancer cell cultures are subject to progression of the cultures. The emulation of the predicted cell functions in vivo is therefore not only determined by the type of culture in vitro but also by the confluence or diameter of the 2D or 3D cultures, respectively. Consequently, the successful implementation of 3D models will require phenotypic characterization to verify the relevance of applying these models for drug development.

Abstract C249: Detection of tissue and bone marrow clearance of systemically engrafted hematologic tumors by ABT-199 and navitoclax using bioluminescent imaging.

Leukemias and lymphomas are characterized by disseminated disease burden in the circulation, lymphoid tissues and bone marrow. Although anti-cancer agents that can effectively target all of these compartments are associated with better prognosis, there is a paucity of established, longitudinal pre-clinical models that can faithfully reproduce the system-wide disease state of these malignancies. To address this limitation, we induced stable expression of the fusion construct of luc2, a firefly luciferase optimized for expression in mammalian cells, and mCherry, a far red fluorescent protein (named LMC), in an acute lymphoblastic leukemia (ALL) cell line, RS4;11, and a mantle cell lymphoma (MCL) cell line, Granta 519. Intravenous (IV) inoculation of these engineered lines in mice results in reliable tumor engraftment of the bone marrow and other tissues. Additionally, we demonstrate that Granta 519, a cell line harvested from a patient with a highly aggressive blastoid lymphoma, invades the central nervous system (CNS) similar to that observed in some MCL patients. Administration of the Bcl-2 inhibitors ABT-199 or navitoclax to established RS4;11-LMC tumor bearing mice significantly reduced tumor load in all compartments. This reflected the clinical activity achieved with navitoclax and, in particular, ABT-199, in recent clinical trials. Further, ABT-199 and navitoclax combined with bendamustine-rituximab (BR) induces significant regression of Granta 519-LMC tumors in the bone marrow and CNS, demonstrating that the Bcl-2 agents can access MCL cells which have invaded the CNS. Taken together, these data demonstrate that IV inoculation of RS4;11-LMC and Granta 519-LMC leads to engraftment of tumor cells in clinically relevant sites. The ability to both model systemic disease and monitor drug treatment in a longitudinal fashion constitutes a translational advantage for the study and development of anticancer therapeutics. Disclosures: SA, AO, JChen, BJC, JClarin, KF, TM, SM, SS, MLS, SKT, JL, AJS, ERB and JH are employees of AbbVie. This study was sponsored by AbbVie. AbbVie contributed to the study design, research and interpretation of data, writing, reviewing and approving the publication. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C249. Citation Format: Scott L. Ackler, Anatol Oleksijew, Jun Chen, Brenda J. Chyla, Jerry Clarin, Kelly Foster, Thomas McGonigal, Sasmita Mishra, Sally Schlessinger, Morey L. Smith, Stephen K. Tahir, Joel Leverson, Andrew J. Souers, Erwin R. Boghaert, Jonathan Hickson. Detection of tissue and bone marrow clearance of systemically engrafted hematologic tumors by ABT-199 and navitoclax using bioluminescent imaging. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C249.