Patricia S. Tsang

The MYCN oncogene is a direct target of miR-34a.

Loss of 1p36 heterozygosity commonly occurs with MYCN amplification in neuroblastoma tumors, and both are associated with an aggressive phenotype. Database searches identified five microRNAs that map to the commonly deleted region of 1p36 and we hypothesized that the loss of one or more of these microRNAs contributes to the malignant phenotype of MYCN-amplified tumors. By bioinformatic analysis, we identified that three out of the five microRNAs target MYCN and of these miR-34a caused the most significant suppression of cell growth through increased apoptosis and decreased DNA synthesis in neuroblastoma cell lines with MYCN amplification. Quantitative RT–PCR showed that neuroblastoma tumors with 1p36 loss expressed lower level of miR-34a than those with normal copies of 1p36. Furthermore, we demonstrated that MYCN is a direct target of miR-34a. Finally, using a series of mRNA expression profiling experiments, we identified other potential direct targets of miR-34a, and pathway analysis demonstrated that miR-34a suppresses cell-cycle genes and induces several neural-related genes. This study demonstrates one important regulatory role of miR-34a in cell growth and MYCN suppression in neuroblastoma.

Identification of FGFR4-activating mutations in human rhabdomyosarcomas that promote metastasis in xenotransplanted models

Rhabdomyosarcoma (RMS) is a childhood cancer originating from skeletal muscle, and patient survival is poor in the presence of metastatic disease. Few determinants that regulate metastasis development have been identified. The receptor tyrosine kinase FGFR4 is highly expressed in RMS tissue, suggesting a role in tumorigenesis, although its functional importance has not been defined. Here, we report the identification of mutations in FGFR4 in human RMS tumors that lead to its activation and present evidence that it functions as an oncogene in RMS. Higher FGFR4 expression in RMS tumors was associated with advanced-stage cancer and poor survival, while FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. Moreover, 6 FGFR4 tyrosine kinase domain mutations were found among 7 of 94 (7.5%) primary human RMS tumors. The mutants K535 and E550 increased autophosphorylation, Stat3 signaling, tumor proliferation, and metastatic potential when expressed in a murine RMS cell line. These mutants also transformed NIH 3T3 cells and led to an enhanced metastatic phenotype. Finally, murine RMS cell lines expressing the K535 and E550 FGFR4 mutants were substantially more susceptible to apoptosis in the presence of a pharmacologic FGFR inhibitor than the control cell lines expressing the empty vector or wild-type FGFR4. Together, our results demonstrate that mutationally activated FGFR4 acts as an oncogene, and these are what we believe to be the first known mutations in a receptor tyrosine kinase in RMS. These findings support the potential therapeutic targeting of FGFR4 in RMS.

Respiratory Flexibility in Response to Inhibition of Cytochrome c Oxidase in Mycobacterium tuberculosis

ABSTRACT We report here a series of five chemically diverse scaffolds that have in vitro activities on replicating and hypoxic nonreplicating bacilli by targeting the respiratory bc1 complex in Mycobacterium tuberculosis in a strain-dependent manner. Deletion of the cytochrome bd oxidase generated a hypersusceptible mutant in which resistance was acquired by a mutation in qcrB. These results highlight the promiscuity of the bc1 complex and the risk of targeting energy metabolism with new drugs.

screening a panel of drugs with diverse mechanisms of action yields potential therapeutic agents against neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Despite current aggressive therapy, the survival rate for high risk NB remains less than 40%. To identify novel effective chemo-agents against NB, we screened a panel of 96 drugs against two NB cell lines, SK-N-AS and SH-SY5Y. We found 30 compounds that were active against NB cell lines at ≤ 10 μM concentration. More interestingly, 17 compounds are active at ≤ 1 μM concentration, and they act through a wide spectrum of diverse mechanisms such as mitotic inhibition, topoisomerase inhibition, targeting various biological pathways, and unknown mechanisms. The majority of these active compounds also induced caspase 3/7 by more than 2-fold. Of these 17 active compounds against NB cell lines at sub-micromolar concentration, 11 compounds are not currently used to treat NB. Among them, 9 are FDA approved compounds, and 3 agents are undergoing clinical trials for various malignancies. Furthermore, we identified 4 agents active against these NB cell lines that have not yet been tested in the clinical setting. Finally we demonstrated that Cucurbitacin I inhibits neuroblastoma cell growth through inhibition of STAT3 pathway. These drugs thus represent potential novel therapeutic agents for patients with NB, and further validation studies are needed to translate them to the clinic.

Synthetic Lethal Screen Identifies NF-κB as a Target for Combination Therapy with Topotecan for patients with Neuroblastoma

BackgroundDespite aggressive multimodal treatments the overall survival of patients with high-risk neuroblastoma remains poor. The aim of this study was to identify novel combination chemotherapy to improve survival rate in patients with high-risk neuroblastoma.MethodsWe took a synthetic lethal approach using a siRNA library targeting 418 apoptosis-related genes and identified genes and pathways whose inhibition synergized with topotecan. Microarray analyses of cells treated with topotecan were performed to identify if the same genes or pathways were altered by the drug. An inhibitor of this pathway was used in combination with topotecan to confirm synergism by in vitro and in vivo studies.ResultsWe found that there were nine genes whose suppression synergized with topotecan to enhance cell death, and the NF-κB signaling pathway was significantly enriched. Microarray analysis of cells treated with topotecan revealed a significant enrichment of NF-κB target genes among the differentially altered genes, suggesting that NF-κB pathway was activated in the treated cells. Combination of topotecan and known NF-κB inhibitors (NSC 676914 or bortezomib) significantly reduced cell growth and induced caspase 3 activity in vitro. Furthermore, in a neuroblastoma xenograft mouse model, combined treatment of topotecan and bortezomib significantly delayed tumor formation compared to single-drug treatments.ConclusionsSynthetic lethal screening provides a rational approach for selecting drugs for use in combination therapy and warrants clinical evaluation of the efficacy of the combination of topotecan and bortezomib or other NF-κB inhibitors in patients with high risk neuroblastoma.

Preparation and Evaluation of Potent Pentafluorosulfanyl-Substituted Anti-Tuberculosis Compounds

The global fight to stop tuberculosis (TB) remains a great challenge, particularly with the increase in drug‐resistant strains and a lack of funding to support the development of new treatments. To bolster a precarious drug pipeline, we prepared a focused panel of eight pentafluorosulfanyl (SF5) compounds which were screened for their activity against Mycobacterium tuberculosis (Mtb) H37Rv in three different assay conditions and media. All eight compounds had sub‐micromolar potency, and four displayed MICs <100 nm. Seven compounds were evaluated against non‐replicating and mono‐drug‐resistant Mtb, and for their ability to inhibit Mtb within the macrophage. The greatest potency was observed against intracellular Mtb (MIC <10 nm for three compounds), which is often the most challenging to target. In general, the SF5‐bearing compounds were very similar to their CF3 counterparts, with the major differences observed being their in vitro ADME properties. Two SF5‐bearing compounds were found to have greater protein binding than their corresponding CF3 counterparts, but were also less metabolized in human microsomes, resulting in longer half‐lives.

MEPicides: potent antimalarial prodrugs targeting isoprenoid biosynthesis

The emergence of Plasmodium falciparum resistant to frontline therapeutics has prompted efforts to identify and validate agents with novel mechanisms of action. MEPicides represent a new class of antimalarials that inhibit enzymes of the methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, including the clinically validated target, deoxyxylulose phosphate reductoisomerase (Dxr). Here we describe RCB-185, a lipophilic prodrug with nanomolar activity against asexual parasites. Growth of P. falciparum treated with RCB-185 was rescued by isoprenoid precursor supplementation, and treatment substantially reduced metabolite levels downstream of the Dxr enzyme. In addition, parasites that produced higher levels of the Dxr substrate were resistant to RCB-185. Notably, environmental isolates resistant to current therapies remained sensitive to RCB-185, the compound effectively treated sexually-committed parasites, and was both safe and efficacious in malaria-infected mice. Collectively, our data demonstrate that RCB-185 potently and selectively inhibits Dxr in P. falciparum, and represents a promising lead compound for further drug development.

Correction for Arora et al., “Respiratory Flexibility in Response to Inhibition of Cytochrome c Oxidase in Mycobacterium tuberculosis”

Volume 58, no. 11, p. 6962–6965, 2014, [https://doi.org/10.1128/AAC.03486-14][1]. Page 6963, left column, second paragraph: the following text in lines 5 to 10 should be deleted. “We deleted this oxidase in H37Rv by replacing a 221-bp MluI fragment in the cydABDC operon with the aph gene

Abstract 3982: Super-resolution imaging of drug delivery to live cells using real-time structured illumination microscopy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Imaging the effects of drug delivery in live cells is most often performed by confocal microscopy, which has a resolution limited by the diffraction of light. Here, we report the development of a novel method for imaging drug delivery to live cells using real-time super-resolution microscopy in wide-field mode. Structured illumination microscopy (SIM) is one of several super-resolving microscopy techniques and is capable of imaging beyond the resolution of conventional microscopy to less than 100 nm. In SIM, each super-resolution (SR) image is computationally constructed from a dataset of numerous raw images. Due to the computational time required for SR image construction, datasets are usually post-processed to avoid slowdowns during the acquisition process. Although post-processing is convenient, this approach has limited use since SR images are not available during image acquisition and thus cannot be utilized for real-time evaluation of the sample. Utilizing SR images in real-time is particularly important when objects of interest are smaller than the resolution limit of conventional microscopy. Applications such as determining optimal focus and finding desirable regions of interest are depending on real-time feedback to user. When performing real-time SIM imaging live cells, image acquisition, processing, and the display of SR images must occur in rapid succession thereby reducing the effects of molecular movement. To achieve the data processing speed necessary for real-time imaging of live cells, we utilized a graphics processing unit (GPU) to perform high-speed SR image construction. Each function for processing SIM data was optimized for parallel operation using GPU kernels. Excluding data transfer overhead, the actual GPU processing time for synthesizing the SR image was 45 milliseconds. Compared with parallel processing on a multi-core computer (CPU), using the GPU achieved a 45-fold increase in processing speed. Overall, data throughput using the GPU attained an 8-fold speed increase over CPU processing, thus allowing real-time high resolution imaging. We applied this SIM platform to record the action of nocodazole on microtubule depolymerization in HeLa cells. We are currently developing this real-time imaging method to investigate the delivery and release mechanism of therapeutic cargoes contained within nanoparticles. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3982.