Noelle S. Williams

Small molecule–mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer

SUMMARY The pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two novel classes of small molecules that disrupt Wnt pathway responses - whereas one class inhibits the activity of Porcupine (Porcn), a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, suppressors of Wnt/β-catenin pathway activity. With these small molecules we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/β-catenin pathway response in vivo, and establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

Targeting QseC Signaling and Virulence for Antibiotic Development

Many bacterial pathogens rely on a conserved membrane histidine sensor kinase, QseC, to respond to host adrenergic signaling molecules and bacterial signals in order to promote the expression of virulence factors. Using a high-throughput screen, we identified a small molecule, LED209, that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. LED209 is not toxic and does not inhibit pathogen growth; however, this compound markedly inhibits the virulence of several pathogens in vitro and in vivo in animals. Inhibition of signaling offers a strategy for the development of broad-spectrum antimicrobial drugs.

Discovery of a Proneurogenic, Neuroprotective Chemical

An in vivo screen was performed in search of chemicals capable of enhancing neuron formation in the hippocampus of adult mice. Eight of 1000 small molecules tested enhanced neuron formation in the subgranular zone of the dentate gyrus. Among these was an aminopropyl carbazole, designated P7C3, endowed with favorable pharmacological properties. In vivo studies gave evidence that P7C3 exerts its proneurogenic activity by protecting newborn neurons from apoptosis. Mice missing the gene encoding neuronal PAS domain protein 3 (NPAS3) are devoid of hippocampal neurogenesis and display malformation and electrophysiological dysfunction of the dentate gyrus. Prolonged administration of P7C3 to npas3(-/-) mice corrected these deficits by normalizing levels of apoptosis of newborn hippocampal neurons. Prolonged administration of P7C3 to aged rats also enhanced neurogenesis in the dentate gyrus, impeded neuron death, and preserved cognitive capacity as a function of terminal aging. PAPERCLIP:

Identification of NF-κB-regulated genes induced by TNFα utilizing expression profiling and RNA interference

Tumor necrosis factor α (TNFα) is a proinflammatory cytokine with important roles in regulating inflammatory responses as well as cell cycle proliferation and apoptosis. Although TNFα stimulates apoptosis, it also activates the transcription factor NF-κB, and studies have shown that inhibition of NF-κB potentiates the cytotoxicity of TNFα. Since several chemotherapy agents act like TNFα to both promote apoptosis and activate NF-κB, understanding the role of NF-κB in suppressing apoptosis may have significant clinical applications. To understand the effects of stimulation with TNFα and the role of NF-κB in regulating this response, a 23k human cDNA microarray was used to screen TNFα-inducible genes in HeLa cells. Real-time PCR verified expression changes in 16 of these genes and revealed three distinct temporal patterns of expression after TNFα stimulation. Using RNA interference to disrupt expression of the p65 subunit of NF-κB, all but two of the genes were shown to depend on this transcription factor for their expression, which correlated well with the existence of NF-κB binding sites in most of their promoters. Inflammatory, proapoptotic, and antiapoptotic genes were all shown to be regulated by NF-κB, demonstrating the wide variety of targets activated by NF-κB signaling and the necessity of differentiating among these genes for therapeutic purposes.

Natural killer cell differentiation: insights from knockout and transgenic mouse models and in vitro systems

Summary: In the last few years, the routine development of knockout and transgenic mice and the ease with which rare progenitor populations can be isolated from hematopoietic organs and cultured in vitro has facilitated significant advances in understanding the lineage and development of natural killer (NK) cells. Fluorescence‐activated cell sorter analyses have identified a common lymphoid progenitor capable of giving rise to NK, T, and B cells, confirming the lymphoid origin of NK cells. Knockout and transgenic mouse models have pointed to an absolutely critical role for signals sent through the interleukin (IL)‐2/lS receptor β (CD 122) chain and common γ (γc) chain for NK development. Such signals are likely relayed inside the cell by the tyrosine kinase Jak3, which associates with γc. Recently developed IL‐15 and IL‐15 receptor a knockout mice have pinpointed IL‐15 as the mediator of this signal. Other mouse models have indicated an unexpected role for flt3 ligand in early NK‐cell development as well as minor roles for stem cell factor and IL‐7 in expanding NK‐cell progenitor numbers. Finally, in vitro culture systems have proven useful in identifying the point in NK development at which each of these signals is critical.

Development of Proneurogenic, Neuroprotective Small Molecules

Degeneration of the hippocampus is associated with Alzheimers disease and occurs very early in the progression of the disease. Current options for treating the cognitive symptoms associated with Alzheimers are inadequate, giving urgency to the search for novel therapeutic strategies. Pharmacologic agents that safely enhance hippocampal neurogenesis may provide new therapeutic approaches. We discovered the first synthetic molecule, named P7C3, which protects newborn neurons from apoptotic cell death, and thus promotes neurogenesis in mice and rats in the subgranular zone of the hippocampal dentate gyrus, the site of normal neurogenesis in adult mammals. We describe the results of a medicinal chemistry campaign to optimize the potency, toxicity profile, and stability of P7C3. Systematic variation of nearly every position of the lead compound revealed elements conducive toward increases in activity and regions subject to modification. We have discovered compounds that are orally available, nontoxic, stable in mice, rats, and cell culture, and capable of penetrating the blood-brain barrier. The most potent compounds are active at nanomolar concentrations. Finally, we have identified derivatives that may facilitate mode-of-action studies through affinity chromatography or photo-cross-linking.

Regulation of VEGF-induced endothelial cell migration by mitochondrial reactive oxygen species

Endothelial migration is a crucial aspect of a variety of physiologic and pathologic conditions including atherosclerosis and vascular repair. Reactive oxygen species (ROS) function as second messengers during endothelial migration. Multiple intracellular sources of ROS are regulated by cellular context, external stimulus, and the microenvironment. However, the predominant source of ROS during endothelial cell (EC) migration and the mechanisms by which ROS regulate cell migration are incompletely understood. In this study, we tested the hypothesis that mitochondria-derived ROS (mtROS) regulate EC migration. In cultured human umbilical vein endothelial cells, VEGF increased mitochondrial metabolism, promoted mtROS production, and induced cell migration. Either the targeted mitochondrial delivery of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1.

Structure/Activity Relationship Studies of Small-Molecule Inhibitors of Wnt Response

Suppression of oncogenic Wnt-mediated signaling holds promise as an anti-cancer therapeutic strategy. We previously reported a novel class of small molecules (IWR-1/2, inhibitors of Wnt response) that antagonize Wnt signaling by stabilizing the Axin destruction complex. Herein, we present the results of structure-activity relationship studies of these compounds.

A Validated Tumorgraft Model Reveals Activity of Dovitinib Against Renal Cell Carcinoma

Extensively validated tumorgraft model shows activity of investigational agent dovitinib against renal cell carcinoma. Grafting a Better Cancer Model When it comes to predicting drug responsiveness in cancer patients, the standard mouse models (xenografts) get low marks: Drugs that work in these mice are frequently ineffective in humans. Xenograft models are created by injecting human tumor cell lines—which often acquire new mutations in culture—into immunocompromised mice. The resulting tumors are generally different from the original tumor. Tumorgrafts, instead created by implanting fragments of human tumors directly into mice, are generating new excitement among some researchers. Sivanand et al. now describe and validate a tumorgraft model of renal cell carcinoma (RCC) that shows promise for preclinical drug studies. The scientists implanted small fragments of RCC tumor from 94 patients into mice, placing the fragments under the fibrous capsule that surrounds the kidney. Sixteen stable tumor lines—which could be serially passaged to new mice—were ultimately established. Examination by a clinical pathologist revealed that the tumorgrafts were quite similar histologically to the original tumors; gene expression patterns, DNA copy number changes, and most mutations in protein-coding regions were also preserved. In addition, mice bearing tumorgrafts from patients that developed elevated concentrations of serum calcium similarly developed tumor-induced hypercalcemia. Moreover, tumorgraft growth was inhibited by two drugs (sunitinib, approved for treating RCC, and sirolimus, the active metabolite of an approved RCC drug), but did not respond to a lung cancer drug that is inactive against RCC. Finally, dovitinib, an inhibitor of several growth factor receptors that is being studied in clinical trials, suppressed the tumorgrafts more potently than sunitinib or sirolimus, suggesting that dovitinib would be active against RCC in humans. These results apply more broadly: The tumorgrafts should be useful for testing other targeted drugs preclinically—an important need, given that most anticancer drugs that enter clinical trials fail to gain approval. Most anticancer drugs entering clinical trials fail to achieve approval from the U.S. Food and Drug Administration. Drug development is hampered by the lack of preclinical models with therapeutic predictive value. Herein, we report the development and validation of a tumorgraft model of renal cell carcinoma (RCC) and its application to the evaluation of an experimental drug. Tumor samples from 94 patients were implanted in the kidneys of mice without additives or disaggregation. Tumors from 35 of these patients formed tumorgrafts, and 16 stable lines were established. Samples from metastatic sites engrafted at higher frequency than those from primary tumors, and stable engraftment of primary tumors in mice correlated with decreased patient survival. Tumorgrafts retained the histology, gene expression, DNA copy number alterations, and more than 90% of the protein-coding gene mutations of the corresponding tumors. As determined by the induction of hypercalcemia in tumorgraft-bearing mice, tumorgrafts retained the ability to induce paraneoplastic syndromes. In studies simulating drug exposures in patients, RCC tumorgraft growth was inhibited by sunitinib and sirolimus (the active metabolite of temsirolimus in humans), but not by erlotinib, which was used as a control. Dovitinib, a drug in clinical development, showed greater activity than sunitinib and sirolimus. The routine incorporation of models recapitulating the molecular genetics and drug sensitivities of human tumors into preclinical programs has the potential to improve oncology drug development.

Systematic Identification of Molecular Subtype-Selective Vulnerabilities in Non-Small-Cell Lung Cancer

Context-specific molecular vulnerabilities that arise during tumor evolution represent an attractive intervention target class. However, the frequency and diversity of somatic lesions detected among lung tumors can confound efforts to identify these targets. To confront this challenge, we have applied parallel screening of chemical and genetic perturbations within a panel of molecularly annotated NSCLC lines to identify intervention opportunities tightly linked to molecular response indicators predictive of target sensitivity. Anchoring this analysis on a matched tumor/normal cell model from a lung adenocarcinoma patient identified three distinct target/response-indicator pairings that are represented with significant frequencies (6%-16%) in the patient population. These include NLRP3 mutation/inflammasome activation-dependent FLIP addiction, co-occurring KRAS and LKB1 mutation-driven COPI addiction, and selective sensitivity to a synthetic indolotriazine that is specified by a seven-gene expression signature. Target efficacies were validated in vivo, and mechanism-of-action studies informed generalizable principles underpinning cancer cell biology.