Robert F. Padera

Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers.

Somatic mutations that activate phosphoinositide 3-kinase (PI3K) have been identified in the p110-α catalytic subunit (encoded by PIK3CA). They are most frequently observed in two hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Although the p110-α mutants are transforming in vitro, their oncogenic potential has not been assessed in genetically engineered mouse models. Furthermore, clinical trials with PI3K inhibitors have recently been initiated, and it is unknown if their efficacy will be restricted to specific, genetically defined malignancies. In this study, we engineered a mouse model of lung adenocarcinomas initiated and maintained by expression of p110-α H1047R. Treatment of these tumors with NVP-BEZ235, a dual pan–PI3K and mammalian target of rapamycin (mTOR) inhibitor in clinical development, led to marked tumor regression as shown by positron emission tomography–computed tomography, magnetic resonance imaging and microscopic examination. In contrast, mouse lung cancers driven by mutant Kras did not substantially respond to single-agent NVP-BEZ235. However, when NVP-BEZ235 was combined with a mitogen-activated protein kinase kinase (MEK) inhibitor, ARRY-142886, there was marked synergy in shrinking these Kras-mutant cancers. These in vivo studies suggest that inhibitors of the PI3K-mTOR pathway may be active in cancers with PIK3CA mutations and, when combined with MEK inhibitors, may effectively treat KRAS mutated lung cancers.

BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models

Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.

Biologic activity of cytotoxic T lymphocyte-associated antigen 4 antibody blockade in previously vaccinated metastatic melanoma and ovarian carcinoma patients

A large number of cancer-associated gene products evoke immune recognition, but host reactions rarely impede disease progression. The weak immunogenicity of nascent tumors contributes to this failure in host defense. Therapeutic vaccines that enhance dendritic cell presentation of cancer antigens increase specific cellular and humoral responses, thereby effectuating tumor destruction in some cases. The attenuation of T cell activation by cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) further limits the potency of tumor immunity. In murine systems, the administration of antibodies that block CTLA-4 function inhibits the growth of moderately immunogenic tumors and, in combination with cancer vaccines, increases the rejection of poorly immunogenic tumors, albeit with a loss of tolerance to normal differentiation antigens. To gain a preliminary assessment of the biologic activity of antagonizing CTLA-4 function in humans, we infused a CTLA-4 blocking antibody (MDX-CTLA4) into nine previously immunized advanced cancer patients. MDX-CTLA4 stimulated extensive tumor necrosis with lymphocyte and granulocyte infiltrates in three of three metastatic melanoma patients and the reduction or stabilization of CA-125 levels in two of two metastatic ovarian carcinoma patients previously vaccinated with irradiated, autologous granulocyte–macrophage colony-stimulating factor-secreting tumor cells. MDX-CTLA4 did not elicit tumor necrosis in four of four metastatic melanoma patients previously immunized with defined melanosomal antigens. No serious toxicities directly attributable to the antibody were observed, although five of seven melanoma patients developed T cell reactivity to normal melanocytes. These findings suggest that CTLA-4 antibody blockade increases tumor immunity in some previously vaccinated cancer patients.

LKB1 modulates lung cancer differentiation and metastasis.

Germline mutation in serine/threonine kinase 11 (STK11, also called LKB1) results in Peutz–Jeghers syndrome, characterized by intestinal hamartomas and increased incidence of epithelial cancers. Although uncommon in most sporadic cancers, inactivating somatic mutations of LKB1 have been reported in primary human lung adenocarcinomas and derivative cell lines. Here we used a somatically activatable mutant Kras-driven model of mouse lung cancer to compare the role of Lkb1 to other tumour suppressors in lung cancer. Although Kras mutation cooperated with loss of p53 or Ink4a/Arf (also known as Cdkn2a) in this system, the strongest cooperation was seen with homozygous inactivation of Lkb1. Lkb1-deficient tumours demonstrated shorter latency, an expanded histological spectrum (adeno-, squamous and large-cell carcinoma) and more frequent metastasis compared to tumours lacking p53 or Ink4a/Arf. Pulmonary tumorigenesis was also accelerated by hemizygous inactivation of Lkb1. Consistent with these findings, inactivation of LKB1 was found in 34% and 19% of 144 analysed human lung adenocarcinomas and squamous cell carcinomas, respectively. Expression profiling in human lung cancer cell lines and mouse lung tumours identified a variety of metastasis-promoting genes, such as NEDD9, VEGFC and CD24, as targets of LKB1 repression in lung cancer. These studies establish LKB1 as a critical barrier to pulmonary tumorigenesis, controlling initiation, differentiation and metastasis.

Novel mutant-selective EGFR kinase inhibitors against EGFR T790M

The clinical efficacy of epidermal growth factor receptor (EGFR) kinase inhibitors in EGFR-mutant non-small-cell lung cancer (NSCLC) is limited by the development of drug-resistance mutations, including the gatekeeper T790M mutation. Strategies targeting EGFR T790M with irreversible inhibitors have had limited success and are associated with toxicity due to concurrent inhibition of wild-type EGFR. All current EGFR inhibitors possess a structurally related quinazoline-based core scaffold and were identified as ATP-competitive inhibitors of wild-type EGFR. Here we identify a covalent pyrimidine EGFR inhibitor by screening an irreversible kinase inhibitor library specifically against EGFR T790M. These agents are 30- to 100-fold more potent against EGFR T790M, and up to 100-fold less potent against wild-type EGFR, than quinazoline-based EGFR inhibitors in vitro. They are also effective in murine models of lung cancer driven by EGFR T790M. Co-crystallization studies reveal a structural basis for the increased potency and mutant selectivity of these agents. These mutant-selective irreversible EGFR kinase inhibitors may be clinically more effective and better tolerated than quinazoline-based inhibitors. Our findings demonstrate that functional pharmacological screens against clinically important mutant kinases represent a powerful strategy to identify new classes of mutant-selective kinase inhibitors.

The biocompatibility of mesoporous silicates

Micro- and nano-mesoporous silicate particles are considered potential drug delivery systems because of their ordered pore structures, large surface areas and the ease with which they can be chemically modified. However, few cytotoxicity or biocompatibility studies have been reported, especially when silicates are administered in the quantities necessary to deliver low-potency drugs. The biocompatibility of mesoporous silicates of particle sizes approximately 150 nm, approximately 800 nm and approximately 4 microm and pore sizes of 3 nm, 7 nm and 16 nm, respectively, is examined here. In vitro, mesoporous silicates showed a significant degree of toxicity at high concentrations with mesothelial cells. Following subcutaneous injection of silicates in rats, the amount of residual material decreased progressively over 3 months, with good biocompatibility on histology at all time points. In contrast, intra-peritoneal and intra-venous injections in mice resulted in death or euthanasia. No toxicity was seen with subcutaneous injection of the same particles in mice. Microscopic analysis of the lung tissue of the mice indicated that death may be due to thrombosis. Although local tissue reaction to mesoporous silicates was benign, they caused severe systemic toxicity. This toxicity might be mitigated by modification of the materials.

Human Semilunar Cardiac Valve Remodeling by Activated Cells From Fetus to Adult Implications for Postnatal Adaptation, Pathology, and Tissue Engineering

Background— The evolution of cell phenotypes and matrix architecture in cardiac valves during fetal maturation and postnatal adaptation through senescence remains unexplored. Methods and Results— We hypothesized that valvular interstitial (VIC) and endothelial cell (VEC) phenotypes, critical for maintaining valve function, change throughout life in response to environmental stimuli. We performed quantitative histological assessment of 91 human semilunar valves obtained from fetuses at 14 to 19 and 20 to 39 weeks’ gestation; neonates minutes to 30 days old; children aged 2 to 16 years; and adults. A trilaminar architecture appeared by 36 weeks of gestation but remained rudimentary compared with that of adult valves. VECs expressed an activated phenotype throughout fetal development. VIC density, proliferation, and apoptosis were significantly higher in fetal than adult valves. Pulmonary and aortic fetal VICs showed an activated myofibroblast-like phenotype (&agr;-actin expression), abundant embryonic myosin, and matrix metalloproteinase-collagenases, which indicates an immature/activated phenotype engaged in matrix remodeling versus a quiescent fibroblast-like phenotype in adults. At birth, the abrupt change from fetal to neonatal circulation was associated with a greater number of &agr;-actin–positive VICs in neonatal aortic versus pulmonary valves. Collagen content increased from early to late fetal stages but was subsequently unchanged, whereas elastin significantly increased postnatally. Collagen fiber color analysis revealed a progressive temporal decrease in thin fibers and a corresponding increase in thick fibers. Additionally, collagen fibers were more aligned in adult than fetal valves. Conclusions— Fetal valves possess a dynamic/adaptive structure and contain cells with an activated/immature phenotype. During postnatal life, activated cells gradually become quiescent, whereas collagen matures, which suggests a progressive, environmentally mediated adaptation.

Integration of engineered cartilage.

The structure and function of cartilaginous constructs, engineered in vitro using bovine articular chondrocytes, biodegradable scaffolds and bioreactors, can be modulated by the conditions and duration of tissue cultivation. We hypothesized that the integrative properties of engineered cartilage depend on developmental stage of the construct and the extracellular matrix content of adjacent cartilage, and that some aspects of integration can be studied under controlled in vitro conditions. Disc‐shaped constructs (cultured for 5±1 days or 5±1 weeks) or explants (untreated or trypsin treated cartilage) were sutured into ring‐shaped explants (untreated or trypsin treated cartilage) to form composites that were cultured for an additional 1‐8 weeks in bioreactors and evaluated biochemically, histologically and mechanically (compressive stiffness of the central disk, adhesive strength of the integration interface). Immature constructs had poorer mechanical properties but integrated better than either more mature constructs or cartilage explants. Integration of immature constructs involved cell proliferation and the progressive formation of cartilaginous tissue, in contrast to the integration of more mature constructs or native cartilage which involved only the secretion of extracellular matrix components. Integration patterns correlated with the adhesive strength of the disc‐ring interface, which was markedly higher for immature constructs than for either more mature constructs or cartilage explants. Trypsin treatment of the adjacent cartilage further enhanced the integration of immature constructs.

In vitro generation of osteochondral composites

Osteochondral repair involves the regeneration of articular cartilage and underlying bone, and the development of a well-defined tissue-to-tissue interface. We investigated tissue engineering of three-dimensional cartilage/bone composites based on biodegradable polymer scaffolds, chondrogenic and osteogenic cells. Cartilage constructs were created by cultivating primary bovine calf articular chondrocytes on polyglycolic acid meshes; bone-like constructs were created by cultivating expanded bovine calf periosteal cells on foams made of a blend of poly-lactic-co-glycolic acid and polyethylene glycol. Pairs of constructs were sutured together after 1 or 4 weeks of isolated culture, and the resulting composites were cultured for an additional 4 weeks. All composites were structurally stable and consisted of well-defined cartilaginous and bone-like tissues. The fraction of glycosaminoglycan in the cartilaginous regions increased with time, both in isolated and composite cultures. In contrast, the mineralization in bone-like regions increased during isolated culture, but remained approximately constant during the subsequent composite culture. The integration at the cartilage/bone interface was generally better for composites consisting of immature (1-week) than mature (4-week) constructs. This study demonstrates that osteochondral tissue composites for potential use in osteochondral repair can be engineered in vitro by culturing mammalian chondrocytes and periosteal cells on appropriate polymer scaffolds.

Spatially controlled cell engineering on biodegradable polymer surfaces

Controlling receptor‐mediated interactions between cells and template surfaces is a central principle in many tissue engineering procedures (1–3). Biomaterial surfaces engineered to present cell adhesion ligands undergo integrin‐mediated molecular interactions with cells (1, 4, 5), stimulating cell spreading, and differentiation (6–8). This provides a mechanism for mimicking natural cell‐to‐matrix interactions. Further sophistication in the control of cell interactions can be achieved by fabricating surfaces on which the spatial distribution of ligands is restricted to micron‐scale pattern features (9–14). Patterning technology promises to facilitate spatially controlled tissue engineering with applications in the regeneration of highly organized tissues. These new applications require the formation of ligand patterns on biocompatible and biodegradable templates, which control tissue regeneration processes, before removal by metabolism. We have developed a method of generating micron‐scale patterns of any biotinylated ligand on the surface of a biodegradable block copolymer, polylactide‐poly(ethylene glycol). The technique achieves control of biomolecule deposition with nanometer precision. Spatial control over cell development has been observed when using these templates to culture bovine aortic endothelial cells and PC12 nerve cells. Furthermore, neurite extension on the biodegradable polymer surface is directed by pattern features composed of peptides containing the IKVAV sequence (15, 16), suggesting that directional control over nerve regeneration on biodegradable biomaterials can be achieved.—Patel, N., Padera, R., Sanders, G. H. W., Cannizzaro, S. M., Davies, M. C., Langer, R., Roberts, C. J., Tendler, S. J. B., Williams, P. M., and Shakesheff, K. M. Spatially controlled cell engineering on biodegradable polymer surfaces. FASEB J. 12, 1447–1454 (1998)