Andrew W. McFadden

Identification of the Receptor Tyrosine Kinase c-Met and Its Ligand, Hepatocyte Growth Factor, as Therapeutic Targets in Clear Cell Sarcoma

Clear cell sarcoma (CCS), a childhood tumor of the tendons and aponeuroses, is uniformly fatal once it has metastasized because of its profound therapeutic resistance. CCS is characterized by production of a chimeric transcription factor, EWS-ATF1, which is formed as the result of a disease-specific chromosomal translocation. EWS-ATF1 activates the melanocyte transcription factor MITF, which in turn activates transcription of c-Met, an oncogenic receptor tyrosine kinase recently shown to be activated in CCS. Based on this connection, we hypothesized that c-Met inhibition may offer a strategy to treat CCS, as an indirect tactic to defeat a transforming pathway downstream of EWS-ATF1. Here, we show that primary CCS and CCS-derived cell lines express c-Met, which is activated in an autocrine fashion by its ligand hepatocyte growth factor (HGF)/scatter factor in some CCS cell lines. c-Met expression is critical for CCS invasion, chemotaxis, and survival. Blocking c-Met activity with a small-molecule inhibitor (SU11274) or a neutralizing antibody to its ligand HGF (AMG 102) significantly reduced CCS cell growth in culture. Similarly, AMG 102 significantly suppressed in vivo tumor growth in an autocrine xenograft model of CCS. Collectively, these findings suggest the HGF:c-Met signaling axis as a candidate therapeutic target to improve clinical management of CCS.

CIB1 is an endogenous inhibitor of agonist-induced integrin αIIbβ3 activation

In response to agonist stimulation, the αIIbβ3 integrin on platelets is converted to an active conformation that binds fibrinogen and mediates platelet aggregation. This process contributes to both normal hemostasis and thrombosis. Activation of αIIbβ3 is believed to occur in part via engagement of the β3 cytoplasmic tail with talin; however, the role of the αIIb tail and its potential binding partners in regulating αIIbβ3 activation is less clear. We report that calcium and integrin binding protein 1 (CIB1), which interacts directly with the αIIb tail, is an endogenous inhibitor of αIIbβ3 activation; overexpression of CIB1 in megakaryocytes blocks agonist-induced αIIbβ3 activation, whereas reduction of endogenous CIB1 via RNA interference enhances activation. CIB1 appears to inhibit integrin activation by competing with talin for binding to αIIbβ3, thus providing a model for tightly controlled regulation of αIIbβ3 activation.

Molecular basis of CIB binding to the integrin αIIb cytoplasmic domain

Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the αIIb cytoplasmic tail of the platelet integrin, αIIbβ3. Here we describe regions within CIB and αIIb that interact with one another. CIB binding to αIIb cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of αIIb. This region is analogous to the α-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind αIIb in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to αIIb. The identification of essential interacting regions within αIIb and CIB provides tools for further probing potential interrelated functions of these proteins.

CIB1 is essential for mouse spermatogenesis.

ABSTRACT CIB1 is a 22-kDa calcium binding, regulatory protein with ∼50% homology to calmodulin and calcineurin B. CIB1 is widely expressed and binds to a number of effectors, such as integrin αIIb, PAK1, and polo-like kinases, in different tissues. However, the in vivo functions of CIB1 are not well understood. To elucidate the function of CIB1 in whole animals, we used homologous recombination in embryonic stem cells to generate Cib1−/− mice. Although Cib1−/− mice grow normally, the males are sterile due to disruption of the haploid phase of spermatogenesis. This is associated with reduced testis size and numbers of germ cells in seminiferous tubules, increased germ cell apoptosis, and the loss of elongated spermatids and sperm. Cib1−/− testes also show increased mRNA and protein expression of the cell cycle regulator Cdc2/Cdk1. In addition, mouse embryonic fibroblasts (MEFs) derived from Cib1−/− mice exhibit a much slower growth rate compared to Cib1+/+ MEFs, suggesting that CIB1 regulates the cell cycle, differentiation of spermatogenic germ cells, and/or differentiation of supporting Sertoli cells.

CIB1 Regulates Endothelial Cells and Ischemia-Induced Pathological and Adaptive Angiogenesis

Pathological angiogenesis contributes to various ocular, malignant, and inflammatory disorders, emphasizing the need to understand this process on a molecular level. CIB1 (calcium- and integrin-binding protein), a 22-kDa EF-hand–containing protein, modulates the activity of p21-activated kinase 1 in fibroblasts. Because p21-activated kinase 1 also contributes to endothelial cell function, we hypothesized that CIB1 may have a role in angiogenesis. We found that endothelial cells depleted of CIB1 by either short hairpin RNA or homologous recombination have reduced migration, proliferation, and tubule formation. Moreover, loss of CIB1 in these cells decreases p21-activated kinase 1 activation, downstream extracellular signal-regulated kinase 1/2 activation, and matrix metalloproteinase 2 expression, all of which are known to contribute to angiogenesis. Consistent with these findings, tissues derived from CIB1-deficient (CIB1−/−) mice have reduced growth factor–induced microvessel sprouting in ex vivo organ cultures and in vivo Matrigel plugs. Furthermore, in response to ischemia, CIB1−/− mice demonstrate decreased pathological retinal and adaptive hindlimb angiogenesis. Ischemic CIB1−/− hindlimbs also demonstrate increased tissue damage and significantly reduced p21-activated kinase 1 activation. These data therefore reveal a critical role for CIB1 in ischemia-induced pathological and adaptive angiogenesis.

PTEN deficiency mediates a reciprocal response to IGFI and mTOR inhibition.

Recent evidence implicates the insulin-like growth factor (IGF) pathway in development of Ewing sarcoma, a highly malignant bone and soft-tissue tumor that primarily affects children and young adults. Despite promising results from preclinical studies of therapies that target this pathway, early-phase clinical trials have shown that a significant fraction of patients do not benefit, suggesting that cellular factors determine tumor sensitivity. Using FAIRE-seq, a chromosomal deletion of the PTEN locus in a Ewing sarcoma cell line was identified. In primary tumors, PTEN deficiency was observed in a large subset of cases, although not mediated by large chromosomal deletions. PTEN loss resulted in hyperactivation of the AKT signaling pathway. PTEN rescue led to decreased proliferation, inhibition of colony formation, and increased apoptosis. Strikingly, PTEN loss decreased sensitivity to IGF1R inhibitors but increased responsiveness to temsirolimus, a potent mTOR inhibitor, as marked by induction of autophagy. These results suggest that PTEN is lost in a significant fraction of primary tumors, and this deficiency may have therapeutic consequences by concurrently attenuating responsiveness to IGF1R inhibition while increasing activity of mTOR inhibitors. The identification of PTEN status in the tumors of patients with recurrent disease could help guide the selection of therapies. Implications: PTEN status in Ewing sarcoma affects cellular responses to IGFI and mTOR-directed therapy, thus justifying its consideration as a biomarker in future clinical trials. Mol Cancer Res; 12(11); 1610–20. ©2014 AACR.

Valproic acid reduces the tolerability of temsirolimus in children and adolescents with solid tumors

A pediatric study has established a maximum tolerated dose (MTD) for temsirolimus (Tem) of more than 150 mg/m2 intravenously/week. A phase I trial was conducted to establish the MTD for Tem in combination with valproic acid (VPA) in children and adolescents with refractory solid tumors. The secondary aims included expression of mammalian target of rapamycin (mTOR) markers on archival tumor tissue; Tem pharmacokinetics; assessment of histone acetylation (HA); and tumor response. Patients were treated with VPA (5 mg/kg orally three times daily) with a target serum level of 75–100 mcg/ml. Tem was started at an initial dose of 60 mg/m2/week. Pharmacokinetics and HA measurements were performed during weeks 1 and 5. Two of the first three patients experienced dose-limiting toxicity (grade 3 mucositis). Tem at 35 mg/m2/week was found to be tolerable. Peak Tem concentrations were higher in all patients compared with those in previously published reports of single agent Tem. Increases in HA are correlated with VPA levels. All tumor samples expressed mTORC1 and mTORC2. An objective response was observed in one patient (melanoma), whereas transient stable disease was observed in four other patients (spinal cord ependymoma, alveolar soft part sarcoma, medullary thyroid carcinoma, and hepatocellular carcinoma). The MTD of Tem when administered with VPA is considerably lower than when used as a single agent, with mucositis the major dose-limiting toxicity. The combination merits further study and may have activity in melanoma. Attention to drug–drug interactions will be important in future multiagent trials including Tem.

Bryostatin 1 activates splenic lymphocytes and induces sustained depletion of splenocyte protein kinase C activity in vivo after a single intravenous administration

Bryostatin 1 activates and subsequently down-regulates protein kinase C (PKC) in vitro and has potential use as an immunomodulator and as an anti-cancer agent. Despite extensive examination of Its activities in vitro and anti-tumor effects in vivo, previous studies have failed to document that bryostatin 1 modulates total cellular PKC activity in tumor or normal tissues when administered in vivo. After a single bolus injection of bryostatin 1 (1.0 μg) in normal C57Bl/6 mice, blood was drawn at various intervals and assayed for bryostatin 1 levels. In addition, spleens from bryostatin-treated mice were harvested 10 min to 10 days after treatment, weighed and analyzed for cell numbers, PKC activity and cell surface phenotypes. Bryostatin 1 levels in plasma rose rapidly, reaching peak levels of 56.5 nM less than 1 min after injection, and then declined to undetectable levels by 1 h. A similar pattern was observed when bryostatin 1 was incubated with leukemia cells in vitro, raising the possibility that the rapid fall in plasma levels results from intracellular uptake and binding. Bryostatin 1 induced marked depletion of total splenocyte PKC activity (as much as 69% relative to control values) at 24–96 h after drug administration, but not at earlier times (i.e. 1 h). A single injection of bryostatin 1 also induced expression of the T cell activation marker CD69, leading to positivity in 53% of cells at 3–24 h versus 11% in control mice, and resulted in marked splenomegaly, associated with increased numbers of nucleated cells at 48–96 h. Together, these studies demonstrate that despite rapid disappearance of the drug from plasma, a single i.v. dose of bryostatin 1 exhibits significant and sustained effects on normal murine spleen cells, including early lymphocyte activation, prolonged depletion of PKC activity, spenocyte proliferation and splenomegaly. These findings may have implications for attempts to understand the in vivo effects of bryostatin 1 in normal host tissues.

High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility

Significance Transcriptional regulators lacking enzymatic activity or binding pockets with targetable molecular features have typically been considered “undruggable,” and a reductionist approach based on identification of their molecular targets has largely failed. We have demonstrated that the Ewing sarcoma chimeric transcription factor, EWSR1-FLI1, maintains accessible chromatin at disease-specific regions. We adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), an assay for accessible chromatin, to screen an epigenetically targeted small molecule library for compounds that reverse the disease-associated signature. This approach can be applied broadly for discovery of chromatin-based developmental therapeutics and offers significant advantages because it does not require the selection of a single molecular target. Using this approach, we identified a specific class of compounds with therapeutic potential. Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways.

Polybromo-1 (PBRM1) bromodomains variably influence nucleosome interactions and cellular function

Chromatin remodelers use bromodomains (BDs) to recognize histones. Polybromo 1 (PBRM1 or BAF180) is hypothesized to function as the nucleosome-recognition subunit of the PBAF chromatin-remodeling complex and is frequently mutated in clear cell renal cell carcinoma (ccRCC). Previous studies have applied in vitro methods to explore the binding specificities of the six individual PBRM1 BDs. However, BD targeting to histones and the influence of neighboring BD on nucleosome recognition have not been well characterized. Here, using histone microarrays and intact nucleosomes to investigate the histone-binding characteristics of the six PBRM1 BDs individually and combined, we demonstrate that BD2 and BD4 of PBRM1 mediate binding to acetylated histone peptides and to modified recombinant and cellular nucleosomes. Moreover, we show that neighboring BDs variably modulate these chromatin interactions, with BD1 and BD5 enhancing nucleosome interactions of BD2 and BD4, respectively, whereas BD3 attenuated these interactions. We also found that binding pocket missense mutations in BD4 observed in ccRCC disrupt PBRM1–chromatin interactions and that these mutations in BD4, but not similar mutations in BD2, in the context of full-length PBRM1, accelerate ccRCC cell proliferation. Taken together, our biochemical and mutational analyses have identified BD4 as being critically important for maintaining proper PBRM1 function and demonstrate that BD4 mutations increase ccRCC cell growth. Because of the link between PBRM1 status and sensitivity to immune checkpoint inhibitor treatment, these data also suggest the relevance of BD4 as a potential clinical target.