ABSTRACT
PURPOSE: Plexiform neurofibromas (PNF) are peripheral nerve sheath tumors that cause significant morbidity in persons with neurofibromatosis type 1 (NF1), yet treatment options remain limited. To identify novel therapeutic targets for PNF, we applied an integrated multi-omic approach to quantitatively profile kinome enrichment in a mouse model that has predicted therapeutic responses in clinical trials for NF1-associated PNF with high fidelity. EXPERIMENTAL DESIGN: Utilizing RNA sequencing combined with chemical proteomic profiling of the functionally enriched kinome using multiplexed inhibitor beads coupled with mass spectrometry, we identified molecular signatures predictive of response to CDK4/6 and RAS/MAPK pathway inhibition in PNF. Informed by these results, we evaluated the efficacy of the CDK4/6 inhibitor, abemaciclib, and the ERK1/2 inhibitor, LY3214996, alone and in combination in reducing PNF tumor burden in Nf1flox/flox;PostnCre mice. RESULTS: Converging signatures of CDK4/6 and RAS/MAPK pathway activation were identified within the transcriptome and kinome that were conserved in both murine and human PNF. We observed robust additivity of the CDK4/6 inhibitor, abemaciclib, in combination with the ERK1/2 inhibitor, LY3214996, in murine and human NF1(Nf1) mutant Schwann cells. Consistent with these findings, the combination of abemaciclib (CDK4/6i) and LY3214996 (ERK1/2i) synergized to suppress molecular signatures of MAPK activation and exhibited enhanced antitumor activity in Nf1flox/flox;PostnCre mice in vivo. CONCLUSIONS: These findings provide rationale for the clinical translation of CDK4/6 inhibitors alone and in combination with therapies targeting the RAS/MAPK pathway for the treatment of PNF and other peripheral nerve sheath tumors in persons with NF1.
Subject(s)
Nerve Sheath Neoplasms , Neurofibroma, Plexiform , Neurofibroma , Neurofibromatosis 1 , Humans , Mice , Animals , Neurofibroma, Plexiform/etiology , Neurofibroma, Plexiform/genetics , Neurofibromatosis 1/drug therapy , Neurofibromatosis 1/genetics , MAP Kinase Signaling System , Proteomics , Nerve Sheath Neoplasms/drug therapy , Nerve Sheath Neoplasms/genetics , Protein Kinase Inhibitors/pharmacology , Neurofibroma/complications , Cyclin-Dependent Kinase 4/geneticsABSTRACT
RAS gene mutations are the most frequent oncogenic event in lung cancer. They activate multiple RAS-centric signaling networks among them the MAPK, PI3K, and RB pathways. Within the MAPK pathway, ERK1/2 proteins exert a bottleneck function for transmitting mitogenic signals and activating cytoplasmic and nuclear targets. In view of disappointing antitumor activity and toxicity of continuously applied MEK inhibitors in patients with KRAS-mutant lung cancer, research has recently focused on ERK1/2 proteins as therapeutic targets and on ERK inhibitors for their ability to prevent bypass and feedback pathway activation. Here, we show that intermittent application of the novel and selective ATP-competitive ERK1/2 inhibitor LY3214996 exerts single-agent activity in patient-derived xenograft (PDX) models of RAS-mutant lung cancer. Combination treatments were well tolerated and resulted in synergistic (ERKi plus PI3K/mTORi LY3023414) and additive (ERKi plus CDK4/6i abemaciclib) tumor growth inhibition in PDX models. Future clinical trials are required to investigate if intermittent ERK inhibitor-based treatment schedules can overcome toxicities observed with continuous MEK inhibition and-equally important-to identify biomarkers for patient stratification.
Subject(s)
Genes, ras/drug effects , Lung Neoplasms/drug therapy , Oncogenes/genetics , Protein Kinase Inhibitors/therapeutic use , Cell Line, Tumor , Humans , Lung Neoplasms/pathology , Protein Kinase Inhibitors/pharmacologyABSTRACT
Metastasis is the primary cause of cancer mortality. The primary tumors of colorectal cancer (CRC) often metastasize to the liver. In this study, we have collected 122 samples from 45 CRC patients. Among them, 32 patients have primary tumors, adjacent normal tissues, and matched liver metastases. Thirteen patients have primary tumors without distant metastasis and matched normal tissues. Characterization of these samples was conducted by whole-exome and RNA sequencing and SNP6.0 analysis. Our results revealed no significant difference in genetic alterations including common oncogenic mutations, whole genome mutations and copy number variations between primary and metastatic tumors. We then assembled gene co-expression networks and identified metastasis-correlated gene networks of immune-suppression, epithelial-mesenchymal transition (EMT) and angiogenesis as the key events and potentially synergistic drivers associated with CRC metastasis. Further independent cohort validation using published datasets has verified that these specific gene networks are up regulated throughout the tumor progression. The gene networks of EMT, angiogenesis, immune-suppression and T cell exhaustion are closely correlated with the poor patient outcome and intrinsic anti-PD-1 resistance. These results offer insights of combinational strategy for the treatment of metastatic CRC.
Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/pathology , Epithelial-Mesenchymal Transition , Liver Neoplasms/secondary , Mutation , Neovascularization, Pathologic , Tumor Microenvironment/immunology , Cohort Studies , Colorectal Neoplasms/blood supply , Colorectal Neoplasms/genetics , DNA Copy Number Variations , Follow-Up Studies , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Liver Neoplasms/blood supply , Liver Neoplasms/genetics , Prognosis , Survival Rate , Tumor Microenvironment/geneticsABSTRACT
We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.
Subject(s)
Apoptosis/genetics , Carcinoma, Pancreatic Ductal/genetics , MAP Kinase Signaling System/genetics , Pancreatic Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Apoptosis/drug effects , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Humans , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinase Kinases/metabolism , Mutation/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins p21(ras)/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Pancreatic NeoplasmsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
The ERK pathway is critical in oncogenesis; aberrations in components of this pathway are common in approximately 30% of human cancers. ERK1/2 (ERK) regulates cell proliferation, differentiation, and survival and is the terminal node of the pathway. BRAF- and MEK-targeted therapies are effective in BRAF V600E/K metastatic melanoma and lung cancers; however, responses are short-lived due to emergence of resistance. Reactivation of ERK signaling is central to the mechanisms of acquired resistance. Therefore, ERK inhibition provides an opportunity to overcome resistance and leads to improved efficacy. In addition, KRAS-mutant cancers remain an unmet medical need in which ERK inhibitors may provide treatment options alone or in combination with other agents. Here, we report identification and activity of LY3214996, a potent, selective, ATP-competitive ERK inhibitor. LY3214996 treatment inhibited the pharmacodynamic biomarker, phospho-p90RSK1, in cells and tumors, and correlated with LY3214996 exposures and antitumor activities. In in vitro cell proliferation assays, sensitivity to LY3214996 correlated with ERK pathway aberrations. LY3214996 showed dose-dependent tumor growth inhibition and regression in xenograft models harboring ERK pathway alterations. Importantly, more than 50% target inhibition for up to 8 to 16 hours was sufficient for significant tumor growth inhibition as single agent in BRAF- and KRAS-mutant models. LY3214996 also exhibited synergistic combination benefit with a pan-RAF inhibitor in a KRAS-mutant colorectal cancer xenograft model. Furthermore, LY3214996 demonstrated antitumor activity in BRAF-mutant models with acquired resistance in vitro and in vivo. Based on these preclinical data, LY3214996 has advanced to an ongoing phase I clinical trial (NCT02857270).
Subject(s)
Neoplasms/drug therapy , Precision Medicine , Animals , Disease Models, Animal , Female , Humans , Mice , Mice, Nude , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic useABSTRACT
Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.
Subject(s)
Antineoplastic Agents/therapeutic use , Aurora Kinase A/antagonists & inhibitors , Mitosis/drug effects , Antineoplastic Agents/pharmacology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Female , HeLa Cells , Humans , MaleABSTRACT
The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.
Subject(s)
Computational Biology , Gene Amplification , Genomics , Neoplasms/genetics , Oncogenes/genetics , Cell Transformation, Neoplastic/genetics , Chromosome Mapping , Computational Biology/methods , Databases, Nucleic Acid , Datasets as Topic , Epigenesis, Genetic , Gene Dosage , Gene Expression Regulation, Neoplastic , Genomics/methods , Humans , Intracellular Signaling Peptides and Proteins , MAP Kinase Signaling System , Neoplasms/drug therapy , Proteins , Proto-Oncogene Proteins/geneticsABSTRACT
The phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway is frequently activated in human cancers, and mTOR is a clinically validated target. mTOR forms two distinct multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, metabolism, proliferation, and survival. Rapamycin and its analogues partially inhibit mTOR through allosteric binding to mTORC1, but not mTORC2, and have shown clinical utility in certain cancers. Here, we report the preclinical characterization of OSI-027, a selective and potent dual inhibitor of mTORC1 and mTORC2 with biochemical IC(50) values of 22 nmol/L and 65 nmol/L, respectively. OSI-027 shows more than 100-fold selectivity for mTOR relative to PI3Kα, PI3Kß, PI3Kγ, and DNA-PK. OSI-027 inhibits phosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 as well as the mTORC2 substrate AKT in diverse cancer models in vitro and in vivo. OSI-027 and OXA-01 (close analogue of OSI-027) potently inhibit proliferation of several rapamycin-sensitive and -insensitive nonengineered and engineered cancer cell lines and also, induce cell death in tumor cell lines with activated PI3K-AKT signaling. OSI-027 shows concentration-dependent pharmacodynamic effects on phosphorylation of 4E-BP1 and AKT in tumor tissue with resulting tumor growth inhibition. OSI-027 shows robust antitumor activity in several different human xenograft models representing various histologies. Furthermore, in COLO 205 and GEO colon cancer xenograft models, OSI-027 shows superior efficacy compared with rapamycin. Our results further support the important role of mTOR as a driver of tumor growth and establish OSI-027 as a potent anticancer agent. OSI-027 is currently in phase I clinical trials in cancer patients.
Subject(s)
Imidazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Proteins/antagonists & inhibitors , Sirolimus/pharmacology , Transcription Factors/antagonists & inhibitors , Triazines/pharmacology , Animals , Apoptosis/drug effects , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Enzyme Activation/drug effects , Female , HeLa Cells , Humans , Imidazoles/chemistry , Imidazoles/pharmacokinetics , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Nude , Multiprotein Complexes , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins c-akt/metabolism , TOR Serine-Threonine Kinases , Triazines/chemistry , Triazines/pharmacokinetics , Xenograft Model Antitumor AssaysABSTRACT
The discovery and optimization of a series of imidazo[1,5-a]pyrazine inhibitors of mTOR is described. HTS hits were optimized for potency, selectivity and metabolic stability to provide the orally bioavailable proof of concept compound 4c that demonstrated target inhibition in vivo and concomitant inhibition of tumor growth in an MDA-MB-231 xenograft model.
Subject(s)
Imidazoles/pharmacology , Proteins/antagonists & inhibitors , Pyrazines/pharmacology , Transcription Factors/antagonists & inhibitors , Administration, Oral , Cell Line, Tumor , Humans , Imidazoles/administration & dosage , Mechanistic Target of Rapamycin Complex 1 , Models, Molecular , Multiprotein Complexes , Pyrazines/administration & dosage , TOR Serine-Threonine Kinases , Xenograft Model Antitumor AssaysABSTRACT
The PI3K/Akt/mTOR pathway is frequently activated in human cancers, and mTOR is a clinically validated target for therapeutic intervention in this pathway. The discovery of the involvement of rapamycin-insensitive mTOR complex 2 (mTORC2) in the activation of Akt, combined with the limited clinical antitumor activity of mTOR complex 1 (mTORC1)-directed rapamycin analogs, have led to the discovery of ATP-competitive selective inhibitors of the mTOR kinase that inhibit the function of both mTORC1 and mTORC2. This review describes progress in the identification of selective and novel inhibitors of mTORC1/2, focusing on the profile of inhibitors that are in clinical development.
Subject(s)
Molecular Targeted Therapy , Neoplasms/drug therapy , Proteins/antagonists & inhibitors , Signal Transduction/drug effects , Transcription Factors/antagonists & inhibitors , Animals , Humans , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes , Neoplasms/metabolism , Proteins/metabolism , Signal Transduction/physiology , TOR Serine-Threonine Kinases , Transcription Factors/metabolismABSTRACT
A high-throughput chemiluminescence and ELISA-based biochemical assay to identify mTORC1/mTORC2 kinase inhibitors is described. These mTOR complexes were isolated from HeLa whole cell lysate using mTOR antibodies and in-well immunoprecipitation. The integrity and purity of the mTORC1 and mTORC2 immunocomplexes were confirmed by western blotting. Full-length recombinant 4E-BP1 was used as a substrate and the catalytic activity was measured by detection of p4E-BP1 [T37/46] by a chemiluminescence method. The performance of this assay that can be used to identify dual mTORC1 and mTORC2 kinase inhibitors in a high-throughput 384-well format is described.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Transcription Factors/antagonists & inhibitors , Adenosine Triphosphate/metabolism , Chromones/pharmacology , Drug Evaluation, Preclinical/methods , Enzyme-Linked Immunosorbent Assay , HeLa Cells , Humans , Immunoprecipitation , Indicators and Reagents , Luminescence , Mechanistic Target of Rapamycin Complex 1 , Morpholines/pharmacology , Multiprotein Complexes , Phosphoinositide-3 Kinase Inhibitors , Proteins , Reproducibility of Results , Substrate Specificity , TOR Serine-Threonine KinasesABSTRACT
We previously reported the occurrence of multiple forms of drug metabolizing enzymes in camel tissues. In this study, we demonstrated for the first time, flavin-containing monooxygenase (FMO)-dependent metabolism of two model substrates methimazole (MEM) and N,N'-dimethylaniline (DMA) by camel liver, kidney, brain and intestine. FMO-catalyzed metabolism in the microsomes of camel tissues was independent of cytochrome P450 (CYP) activity and exhibited a pH and temperature dependence characteristic of FMO enzymes. Use of inhibitors of CYP activities, SKF525A, octylamine or antibody against NADPH-P450 reductase, did not significantly alter the FMO-dependent substrate metabolism. Using MEM as a model substrate for FMO activity, we show that camel liver has an activity similar to that in rat and human livers. MEM metabolism in extrahepatic tissues in camels was significantly lower (60%-80%) than that in liver. Our results suggest occurrence of FMO in camel tissues, with catalytic properties similar to those in rat and human livers. These results may help in better understanding the effects of pharmacologically and toxicologically active compounds administered to camels.
Subject(s)
Brain/enzymology , Camelus/metabolism , Intestine, Small/enzymology , Kidney/enzymology , Liver/enzymology , Oxygenases/metabolism , Adult , Aniline Compounds/pharmacology , Animals , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Humans , Male , Methimazole/pharmacology , Microsomes/enzymology , Microsomes, Liver/enzymology , NADP/metabolism , Oxidation-Reduction , Rats , Rats, WistarABSTRACT
CD13/aminopeptidase N (CD13/APN) is a potent regulator of angiogenesis both in vitro and in vivo and transcription of CD13/APN in endothelial cells is induced by angiogenic growth factors via the RAS/MAPK pathway. We have explored the nuclear effectors downstream of this pathway that are responsible for CD13/APN induction. The response to serum/angiogenic growth factors mapped to a 38-bp region of the CD13/APN promoter containing an Ets-core motif that specifically binds a protein complex from nuclear lysates from activated endothelial cells. This motif and the proteins that target it are functionally relevant because mutation of this sequence abrogates CD13/APN transcription. Analysis of endothelial Ets family members showed that Ets-2, and to a lesser extent Ets-1, transactivate CD13/APN promoter activity via the Ets-core motif, whereas Fli, Erg, and NERF are ineffective. We investigated the possibility that the induction of CD13/APN is mediated by phosphorylation of Ets-2 via RAS/MAPK. A phosphorylation-defective Ets-2 mutant, T72A, failed to transactivate CD13/APN, suggesting that Ets-2 phosphorylation is obligatory for CD13/APN induction. To confirm a role for endogenous Ets-2 in CD13/APN expression, we specifically abrogated Ets-2 mRNA and protein by siRNA knockdown that significantly inhibited CD13/APN transcription. Finally, to assess the relevance of Ets-2 in endothelial cell function, we induced endothelial cells containing Ets-2 siRNA oligonucleotides to form capillary networks. Cells containing the Ets-2 inhibitory small interfering RNAs were completely incapable of forming the organized networks characteristic of endothelial morphogenesis. Thus, the phosphorylation of Ets-2 by RAS/MAPK is a prerequisite for CD13/APN endothelial induction and Ets-2 and its targets play essential roles in endothelial cell function.
Subject(s)
CD13 Antigens/genetics , DNA-Binding Proteins , Endothelium, Vascular/metabolism , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Repressor Proteins , Trans-Activators/metabolism , Transcription Factors , Transcription, Genetic/physiology , Cell Line, Tumor , Electrophoretic Mobility Shift Assay , Endothelium, Vascular/cytology , Endothelium, Vascular/enzymology , Humans , Phosphorylation , Proto-Oncogene Protein c-ets-2ABSTRACT
Angiogenesis, the formation of new blood vessels, is a critical step for tumor growth and metastasis and an integral component of the pathologic inflammatory response in arthritis and the proliferative retinopathies. The CD13/aminopeptidase N (CD13/APN) metalloprotease is an important regulator of angiogenesis where its expression on activated blood vessels is induced by angiogenic signals. Here, we show that cytokine induction of CD13/APN in endothelial cells is regulated by distinct Ras effector pathways involving Ras/mitogen-activated protein kinase (MAPK) or PI-3K. Signals transduced by activated Ras, Raf, and mitogen-induced extracellular kinase (MEK) stimulate transcription from the CD13/APN proximal promoter. Inhibition of these pathways and extracellular signal-regulated serine/threonine kinase (ERK-2) and PI-3K by expression of dominant-negative proteins or chemical inhibitors prevented induction of CD13/APN transcription in response to basic fibroblast growth factor (bFGF). We show that Ras-induced signal transduction is required for growth factor-induced angiogenesis, because inhibition of downstream mediators of Ras signaling (MEK or PI-3K) abrogated endothelial cell migration, invasion, and morphogenesis in vitro. Reintroduction of CD13/APN, a shared downstream target of these pathways, overrode the suppressive effect of these inhibitors and restored the function of endothelial cells in migration/invasion and capillary morphogenesis assays. Similarly, inhibition of MEK abrogated cell invasion and the formation of endothelial-lined capillaries in vivo, which was effectively rescued by addition of exogenous CD13/APN protein. These studies provide strong evidence that CD13/APN is an important target of Ras signaling in angiogenesis and is a limiting factor in angiogenic progression.