Yali Kong

A small molecule blocking oncogenic protein EWS-FLI1 interaction with RNA helicase A inhibits growth of Ewing's sarcoma

Many sarcomas and leukemias carry nonrandom chromosomal translocations encoding tumor-specific mutant fusion transcription factors that are essential to their molecular pathogenesis. Ewings sarcoma family tumors (ESFTs) contain a characteristic t(11;22) translocation leading to expression of the oncogenic fusion protein EWS-FLI1. EWS-FLI1 is a disordered protein that precludes standard structure-based small-molecule inhibitor design. EWS-FLI1 binding to RNA helicase A (RHA) is important for its oncogenic function. We therefore used surface plasmon resonance screening to identify compounds that bind EWS-FLI1 and might block its interaction with RHA. YK-4-279, a derivative of the lead compound from the screen, blocks RHA binding to EWS-FLI1, induces apoptosis in ESFT cells and reduces the growth of ESFT orthotopic xenografts. These findings provide proof of principle that inhibiting the interaction of mutant cancer-specific transcription factors with the normal cellular binding partners required for their oncogenic activity provides a promising strategy for the development of uniquely effective, tumor-specific anticancer agents.

High ALDH Activity Identifies Chemotherapy-Resistant Ewing's Sarcoma Stem Cells That Retain Sensitivity to EWS-FLI1 Inhibition

Background Cancer stem cells are a chemotherapy-resistant population capable of self-renewal and of regenerating the bulk tumor, thereby causing relapse and patient death. Ewings sarcoma, the second most common form of bone tumor in adolescents and young adults, follows a clinical pattern consistent with the Cancer Stem Cell model – remission is easily achieved, even for patients with metastatic disease, but relapse remains frequent and is usually fatal. Methodology/Principal Findings We have isolated a subpopulation of Ewings sarcoma cells, from both human cell lines and human xenografts grown in immune deficient mice, which express high aldehyde dehydrogenase (ALDHhigh) activity and are enriched for clonogenicity, sphere-formation, and tumor initiation. The ALDHhigh cells are resistant to chemotherapy in vitro, but this can be overcome by the ATP binding cassette transport protein inhibitor, verapamil. Importantly, these cells are not resistant to YK-4-279, a small molecule inhibitor of EWS-FLI1 that is selectively toxic to Ewings sarcoma cells both in vitro and in vivo. Conclusions/Significance Ewings sarcoma contains an ALDHhigh stem-like population of chemotherapy-resistant cells that retain sensitivity to EWS-FLI1 inhibition. Inhibiting the EWS-FLI1 oncoprotein may prove to be an effective means of improving patient outcomes by targeting Ewings sarcoma stem cells that survive standard chemotherapy.

A boronic acid chalcone analog of combretastatin A-4 as a potent anti-proliferation agent

Chalcones represent a class of natural products that inhibits tubulin assembly. In this study we designed and synthesized boronic acid analogs of chalcones in an effort to compare biological activities with combretastatin A-4, a potent inhibitor of tubulin polymerization. Systematic evaluation of the positional effects of the carbonyl moiety towards inhibition of tubulin polymerization, cancer cell proliferation and angiogenesis revealed that placement of the carbonyl adjacent to the trimethoxybenzene A-ring resulted in more active compounds than when the carbonyl group was placed adjacent to the C-ring. Our study identified a boronic acid chalcone with inhibition towards 16 human cancer cell lines in the 10-200nM range, and another three cell lines with GI(50)-values below 10nM. Furthermore, this drug has significant anti-angiogenesis effects demonstrated by HUVEC tube formation and aortic ring assay.

YK-4-279 Inhibits ERG and ETV1 Mediated Prostate Cancer Cell Invasion

Background Genomic rearrangements involving the ETS family of transcription factors occur in 40–70% of prostate cancer cases. ERG and ETV1 are the most common ETS members observed in these genetic alterations. The high prevalence of these rearrangements and their biological significance represents a novel therapeutic target for the treatment of prostate cancer. Methods and Findings We recently reported the development of YK-4-279, a small molecule inhibitor of EWS-FLI1 oncoprotein in Ewings Sarcoma. Since ERG and ETV1 belong to the same class of ETS factors as FLI1, we tested the ability of YK-4-279 to inhibit biological functions of ERG and ETV1 proteins in prostate cancer. YK-4-279 inhibited ERG and ETV1 mediated transcriptional activity in a luciferase assay. YK-4-279 also decreased ERG and ETV1 downstream target mRNA and protein expression in ETV1-fusion positive LNCaP and ERG fusion positive VCaP cells. YK-4-279 reduced the motility of LNCaP cells in a scratch assay and the invasive phenotype of both LNCaP and VCaP cells in a HUVEC invasion assay. Fusion-negative PC3 cells were unresponsive to YK-4-279. SiRNA mediated ERG knockdown in VCaP cells resulted in a loss of drug responsiveness. Concurrently, transient ERG expression in PC-3 cells resulted in increased invasive potential, which was reduced by YK-4-279. Conclusion These data demonstrate that YK-4-279 inhibits ERG and ETV1 biological activity in fusion-positive prostate cancer cells leading to decreased motility and invasion. Therefore, YK-4-279 may have an impact on metastasis in prostate cancer and it may be further evaluated for its clinical applications in prostate cancer in addition to Ewings sarcoma.

Histone Deacetylase Cytoplasmic Trapping by a Novel Fluorescent HDAC Inhibitor

Inhibitors of histone deacetylases (HDAC) are an important emerging class of drugs for the treatment of cancers. HDAC inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in combinations with chemotherapies and radiation therapy. Although these drugs have important effects on cancer cell growth and functions, the mechanisms underlying HDAC inhibitor activities remain to be fully defined. By using rational drug design, compound 2, a fluorescent class II HDAC targeting inhibitor, was synthesized and observed to accumulate in the cytoplasmic compartments of treated cells, but not in the nuclei. Furthermore, immunostaining of inhibitor exposed cells for HDAC4 showed accumulation of this enzyme in the cytoplasmic compartment with concomitant increased acetylation of tubulin and nuclear histones. These observations support a mechanism by which nuclear histone acetylation is increased as a result of HDAC4 trapping and sequestration in the cytoplasm after binding to compound 2. The HDAC inhibitor offers potential as a novel theranostic agent, combining diagnostic and therapeutic properties in the same molecule. Mol Cancer Ther; 10(9); 1591–9. ©2011 AACR.

Novel carbazole inhibits phospho-STAT3 through induction of protein-tyrosine phosphatase PTPN6.

The aberrant activation of STAT3 occurs in many human cancers and promotes tumor progression. Phosphorylation of a tyrosine at amino acid Y705 is essential for the function of STAT3. Synthesized carbazole derived with fluorophore compound 12 was discovered to target STAT3 phosphorylation. Compound 12 was found to inhibit STAT3-mediated transcription as well as to reduce IL-6 induced STAT3 phosphorylation in cancer cell lines expressing both elevated and low levels of phospho-STAT3 (Y705). Compound 12 potently induced apoptosis in a broad number of TNBC cancer cell lines in vitro and was effective at inhibiting the in vivo growth of human TNBC xenograft tumors (SUM149) without any observed toxicity. Compound 12 also effectively inhibited the growth of human lung tumor xenografts (A549) harboring aberrantly active STAT3. In vitro and in vivo studies showed that the inhibitory effects of 12 on phospho-STAT3 were through up-regulation of the protein-tyrosine phosphatase PTPN6. Our present studies strongly support the continued preclinical evaluation of compound 12 as a potential chemotherapeutic agent for TNBC and cancers with constitutive STAT3 signaling.

A small molecule inhibitor of ETV1, YK-4-279, prevents prostate cancer growth and metastasis in a mouse xenograft model.

Background The erythroblastosis virus E26 transforming sequences (ETS) family of transcription factors consists of a highly conserved group of genes that play important roles in cellular proliferation, differentiation, migration and invasion. Chromosomal translocations fusing ETS factors to promoters of androgen responsive genes have been found in prostate cancers, including the most clinically aggressive forms. ERG and ETV1 are the most commonly translocated ETS proteins. Over-expression of these proteins in prostate cancer cells results in a more invasive phenotype. Inhibition of ETS activity by small molecule inhibitors may provide a novel method for the treatment of prostate cancer. Methods and Findings We recently demonstrated that the small molecule YK-4-279 inhibits biological activity of ETV1 in fusion-positive prostate cancer cells leading to decreased motility and invasion in-vitro. Here, we present data from an in-vivo mouse xenograft model. SCID-beige mice were subcutaneously implanted with fusion-positive LNCaP-luc-M6 and fusion-negative PC-3M-luc-C6 tumors. Animals were treated with YK-4-279, and its effects on primary tumor growth and lung metastasis were evaluated. YK-4-279 treatment resulted in decreased growth of the primary tumor only in LNCaP-luc-M6 cohort. When primary tumors were grown to comparable sizes, YK-4-279 inhibited tumor metastasis to the lungs. Expression of ETV1 target genes MMP7, FKBP10 and GLYATL2 were reduced in YK-4-279 treated animals. ETS fusion-negative PC-3M-luc-C6 xenografts were unresponsive to the compound. Furthermore, YK-4-279 is a chiral molecule that exists as a racemic mixture of R and S enantiomers. We established that (S)-YK-4-279 is the active enantiomer in prostate cancer cells. Conclusion Our results demonstrate that YK-4-279 is a potent inhibitor of ETV1 and inhibits both the primary tumor growth and metastasis of fusion positive prostate cancer xenografts. Therefore, YK-4-279 or similar compounds may be evaluated as a potential therapeutic tool for treatment of human prostate cancer at different stages.

Asymmetric synthesis and evaluation of a hydroxyphenylamide voltage-gated sodium channel blocker in human prostate cancer xenografts

Voltage-gated sodium channels are known to be expressed in neurons and other excitable cells. Recently, voltage-gated sodium channels have been found to be expressed in human prostate cancer cells. α-Hydroxy-α-phenylamides are a new class of small molecules that have demonstrated potent inhibition of voltage-gated sodium channels. The hydroxyamide motif, an isostere of a hydantoin ring, provides an active scaffold from which several potent racemic sodium channel blockers have been derived. With little known about chiral preferences, the development of chiral syntheses to obtain each pure enantiomer for evaluation as sodium channel blockers is important. Using Seebach and Fraters chiral template, cyclocondensation of (R)-3-chloromandelic acid with pivaldehyde furnished both the cis- and trans-2,5-disubsituted dioxolanones. Using this chiral template, we synthesized both enantiomers of 2-(3-chlorophenyl)-2-hydroxynonanamide, and evaluated their ability to functionally inhibit hNa(v) isoforms, human prostate cancer cells and xenograft. Enantiomers of lead demonstrated significant ability to reduce prostate cancer in vivo.

Synthesis and Structure−Activity Relationship Studies of Small Molecule Disruptors of EWS-FLI1 Interactions in Ewing's Sarcoma

EWS-FLI1 is an oncogenic fusion protein implicated in the development of Ewing’s sarcoma family tumors (ESFT). Using our previously reported lead compound 2 (YK-4-279), we designed and synthesized a focused library of analogues. The functional inhibition of the analogues was measured by an EWS-FLI1/NR0B1 reporter luciferase assay and a paired cell screening approach measuring effects on growth inhibition for human cells containing EWS-FLI1 (TC32 and TC71) and control PANC1 cell lines devoid of the oncoprotein. Our data revealed that substitution of electron donating groups at the para-position on the phenyl ring was the most favorable for inhibition of EWS-FLI1 by analogs of 2. Compound 9u (with a dimethylamino substitution) was the most active inhibitor with GI50 = 0.26 ± 0.1 μM. Further, a correlation of growth inhibition (EWS-FLI1 expressing TC32 cells) and the luciferase reporter activity was established (R2 = 0.84). Finally, we designed and synthesized a biotinylated analogue and determined the binding affinity for recombinant EWS-FLI1 (Kd = 4.8 ± 2.6 μM).

Total Synthesis of 7-Hydroxymurrayazolinine, Murrayamine D, and Mahanine via m-Nitro Group Activated Pyran Annulation

The facile total synthesis of the natural product (±)-mahanine was obtained in eight steps with an overall 52% yield from readily accessible known nitrophenol derivative 6. After a one-step, acid-catalyzed annulation, two additional natural products were formed including 7-hydroxymurrayazolinine, representing its first reported total synthesis. In the whole process, the introduction of the m-nitro group significantly enhanced the key pyran annulation reaction through inductive effects.