Highly sensitive proximity mediated immunoassay reveals HER2 status conversion in the circulating tumor cells of metastatic breast cancer patients.

BACKGROUND: The clinical benefits associated with targeted oncology agents are generally limited to subsets of patients. Even with favorable biomarker profiles, many patients do not respond or acquire resistance. Existing technologies are ineffective for treatment monitoring as they provide only static and limited information and require substantial amounts of tissue. Therefore, there is an urgent need to develop methods that can profile potential therapeutic targets with limited clinical specimens during the course of treatment. METHODS: We have developed a novel proteomics-based assay, Collaborative Enzyme Enhanced Reactive-immunoassay (CEER) that can be used for analyzing clinical samples. CEER utilizes the formation of unique immuno-complex between capture-antibodies and two additional detector-Abs on a microarray surface. One of the detector-Abs is conjugated to glucose oxidase (GO), and the other is conjugated to Horse Radish Peroxidase (HRP). Target detection requires the presence of both detector-Abs because the enzyme channeling event between GO and HRP will not occur unless both Abs are in close proximity. RESULTS: CEER was able to detect single-cell level expression and phosphorylation of human epidermal growth factor receptor 2 (HER2) and human epidermal growth factor receptor 1 (HER1) in breast cancer (BCa) systems. The shift in phosphorylation profiles of receptor tyrosine kinases (RTKs) and other signal transduction proteins upon differential ligand stimulation further demonstrated extreme assay specificity in a multiplexed array format. HER2 analysis by CEER in 227 BCa tissues showed superior accuracy when compared to the outcome from immunohistochemistry (IHC) (83% vs. 96%). A significant incidence of HER2 status alteration with recurrent disease was observed via circulating tumor cell (CTC) analysis, suggesting an evolving and dynamic disease progression. HER2-positive CTCs were found in 41% (7/17) while CTCs with significant HER2-activation without apparent over-expression were found in 18% (3/17) of relapsed BCa patients with HER2-negative primary tumors. The apparent 'HER2 status conversion' observed in recurrent BCa may have significant implications on understanding breast cancer metastasis and associated therapeutic development. CONCLUSION: CEER can be multiplexed to analyze pathway proteins in a comprehensive manner with extreme specificity and sensitivity. This format is ideal for analyzing clinical samples with limited availability.

Identification of polymorphisms associated with hypertriglyceridemia and prolonged survival induced by bexarotene in treating non-small cell lung cancer.

BACKGROUND: Bexarotene was evaluated in treating advanced non small cell lung cancer (NSCLC) in two phase III trials. Although a significant survival benefit was not observed for the overall bexarotene-treated population (617 patients), a third of bexarotene-treated patients who developed high-grade hypertriglyceridemia exhibited significantly longer survival. PATIENTS AND METHODS: In order to identify genomic polymorphisms that could serve as potential predictive biomarkers for response and improved survival in NSCLC patients, DNA samples extracted from plasma archived from 403 patients were genotyped using Affymetrix 500K whole genome SNP arrays and/or Sequenom iPLEX™ assays. RESULTS: Fourteen SNPs were identified on nine loci that showed significant associations with high-grade hypertriglyceridemia induced by bexarotene. Four such single nucleotide polymorphisms (SNPs) reside on the region upstream of solute carrier family 10, member 2 (SLC10A2), and one SNP is located close to lymphocyte cytosolic protein 1 (LCP1), whose expression correlated with the activity of bexarotene in tumor cells. CONCLUSION: We identified novel polymorphisms exhibiting significant association with bexarotene induced hypertriglyceridemia, implicating their potential in predicting bexarotene-improved survival response.

Crystal structure of HsEg5 in complex with clinical candidate CK0238273 provides insight into inhibitory mechanism, potency, and specificity.

HsEg5 is an important mitotic kinesin responsible for bipolar spindle formation at early mitosis. A rich body of evidence shows that inhibition of HsEg5 can result in mitotic arrest followed by cellular apoptosis. Recently identified HsEg5 inhibitor, CK0238273, exhibits potent antitumor activity and is currently in clinical trial. Here we report the cocrystal structure of the motor domain of HsEg5 in complex with CK0238273 at a 2.15 A resolution. Compared to the previously published HsEg5-Monastrol complex structure, CK0238273 shares the same induced-fit pocket with similar allosteric inhibitory mechanism. However, CK0238273 shows better fitting to the binding pocket with 65% increase of hydrophobic interaction area than that of Monastrol. Some unique hydrophilic interactions were also observed mostly between the phenyl ring and 8-chloro on quinazolinone of CK0238273 with ARG221 and GLY217. We believe that the combination of these interactions defines the superior potency and specificity of CK0238273.

Bexarotene (LGD1069, Targretin), a selective retinoid X receptor agonist, prevents and reverses gemcitabine resistance in NSCLC cells by modulating gene amplification.

Acquired drug resistance is a major obstacle in cancer therapy. As for many other drugs, this is also the case for gemcitabine, a nucleoside analogue with activity against non-small cell lung cancer (NSCLC). Here, we evaluate the ability of bexarotene to modulate the acquisition and maintenance of gemcitabine resistance in Calu3 NSCLC models. In the prevention model, Calu3 cells treated repeatedly with gemcitabine alone gradually developed resistance. However, with inclusion of bexarotene, the cells remained chemosensitive. RNA analysis showed a strong increase of rrm1 (ribonucleotide reductase M1) expression in the resistant cells (Calu3-GemR), a gene known to be involved in gemcitabine resistance. In addition, the expression of genes surrounding the chromosomal location of rrm1 was increased, suggesting that resistance was due to gene amplification at the chr11 p15.5 locus. Analysis of genomic DNA confirmed that the rrm1 gene copy number was increased over 10-fold. Correspondingly, fluorescence in situ hybridization analysis of metaphase chromosomes showed an intrachromosomal amplification of the rrm1 locus. In the therapeutic model, bexarotene gradually resensitized Calu3-GemR cells to gemcitabine, reaching parental drug sensitivity after 10 treatment cycles. This was associated with a loss in rrm1 amplification. Corresponding with the in vitro data, xenograft tumors generated from the resistant cells did not respond to gemcitabine but were growth inhibited when bexarotene was added to the cytotoxic agent. The data indicate that bexarotene can resensitize gemcitabine-resistant tumor cells by reversing gene amplification. This suggests that bexarotene may have clinical utility in cancers where drug resistance by gene amplification is a major obstacle to successful therapy.

Influence of Bcl-2 family members on the cellular response of small-cell lung cancer cell lines to ABT-737.

ABT-737 is a novel and potent Bcl-2 antagonist with single-agent activity against small-cell lung cancer (SCLC) cell lines. Here, we evaluated the contribution of Bcl-2 family members to the in vitro cellular response of several SCLC cell lines to ABT-737. Relatively higher levels of Bcl-2, Bcl-X(L), Bim and Noxa, and lower levels of Mcl-1 characterized naïve SCLC cell lines that were sensitive to ABT-737. Conversely, a progressive decrease in the relative levels of Bcl-2 and Noxa and a progressive increase in Mcl-1 levels characterized the increased resistance of H146 cells following chronic exposure to ABT-737. Knockdown of Mcl-1 with small interfering RNA sensitized two resistant SCLC cell lines H196 and DMS114 to ABT-737 by enhancing the induction of apoptosis. Likewise, up-regulation of Noxa sensitized H196 cells to ABT-737. Combination treatment with DNA-damaging agents was extremely synergistic with ABT-737 and was associated with the down-regulation of Mcl-1 and the up-regulation of Noxa, Puma, and Bim in H196 cells. Thus, SCLC cells sensitive to ABT-737 expressed the target proteins Bcl-2 and Bcl-X(L), whereas Mcl-1 and factors regulating Mcl-1 function seem to contribute to the overall resistance of SCLC cells to ABT-737. Overall, these observations provide further insight as to the mechanistic bases for ABT-737 efficacy in SCLC and will be helpful for profiling patients and aiding in the rational design of combination therapies.

Studies leading to potent, dual inhibitors of Bcl-2 and Bcl-xL.

Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.

A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo.

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.

Discovery and structure-activity relationship of antagonists of B-cell lymphoma 2 family proteins with chemopotentiation activity in vitro and in vivo.

Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X(L) function, is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-microM binding but were strongly deactivated in the presence of serum. The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of inhibitors bound to both Bcl-X(L) and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-X(L) binding and greatly increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X(L) with a K(i) of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X(L) overexpression against cytokine deprivation in FL5.12 cells with an EC(50) of 0.47 microM. 73R showed little effect on the viability of the human non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model.

Indoloprodigiosins from the C-10 bipyrrolic precursor: new antiproliferative prodigiosin analogs.

The condensation of the C-10 methoxybipyrrole precursor (3) of prodigiosin with indoles and a related pyrrole derivative yields novel analogs of prodigiosin. Biological evaluation of these products revealed compounds that inhibit cancer cell proliferation from 50 nM to 50 microM.

Identification of a small molecule that induces mitotic arrest using a simplified high-content screening assay and data analysis method.

High-content screening has emerged as a new and powerful technique for identifying small-molecule modulators of mammalian cell biology. The authors describe the development and execution of a high-content screen to identify small molecules that induce mitotic arrest in mammalian cancer cells. Many widely used chemotherapeutics, such as Taxol and vinblastine, induce mitotic arrest, and the creation of new drugs that also induce mitotic arrest may have tremendous therapeutic value. In their screen, the authors employed a simple DNA stain (DAPI) and a sensitive nonparametric statistical test to identify compounds from an internal collection of approximately 13,000 high-quality lead-like small molecules. Subsequent analysis of 1 active compound indicated that it induces mitotic arrest, assessed using a high-content phosphohistone H3 detection assay, and caused cell proliferation defects in multiple cancer cell lines. The active compound, a quinazolinone originating from a natural product-like subset of the screened compounds, is active in cells at approximately 500 nM and appears to act by inhibiting the polymerization of tubulin.

Privileged structure-based quinazolinone natural product-templated libraries: identification of novel tubulin polymerization inhibitors.

A focused quinazolinone natural product-templated library was designed and synthesized. Compounds from this privileged structure-based library were identified as antimitotic agents acting through destabilization of tubulin polymerization. The results suggested that 2 could be a privileged substructure.

A highly potent and selective farnesyltransferase inhibitor ABT-100 in preclinical studies.

Ras mutation has been detected in approximately 20-30% of all human carcinomas, primarily in pancreatic, colorectal, lung and bladder carcinomas. The indirect inhibition of Ras activity by inhibiting farnesyltransferase (FTase) function is one therapeutic intervention to control tumor growth. Here we report the preclinical anti-tumor activity of our most advanced FTase inhibitor (FTI), ABT-100, and a direct comparison with the current clinical candidates. ABT-100 is a highly selective, potent and orally bioavailable FTI. It broadly inhibits the growth of solid tumors in preclinical animal models. Thus, ABT-100 is an attractive candidate for further clinical evaluation. In addition, our results provide plausible insights to explain the impressive potency and selectivity of ABT-100. Finally, we have demonstrated that ABT-100 significantly suppresses the expression of vascular endothelial growth factor (VEGF) mRNA and secretion of VEGF protein, as well as inhibiting angiogenesis in the animal model.

Potent farnesyltransferase inhibitor ABT-100 abrogates acute allograft rejection.

BACKGROUND: Farnesyltransferase inhibitors (FTIs) inhibit the function of Ras, a GTPase involved in carcinogenesis and T cell activation. We evaluated the in vitro and in vivo immunomodulatory properties of a rationally designed FTI, ABT-100. METHODS: The effects of ABT-100 on human peripheral blood mononuclear cell (PBMC) proliferation and the expression of the T cell activation markers CD25 and CD69 were studied. In a Wistar to Lewis rat heterotopic cardiac transplant model, ABT-100 was orally dosed alone or with a subtherapeutic course of cyclosporine (CsA). The degree of graft immune cell infiltrate was determined. RESULTS: ABT-100 potently inhibited PBMC proliferation, but did not decrease expression of CD25 and CD69 during activation. Treatment with 25, 50 and 100 mg/kg ABT-100 BID increased allograft mean survival time (MST) to 12.8+/-3 days, 13.5+/-5 days and 13.8+/-3 days, respectively (vs 6.5+/-3 days for controls, p<0.001 by log rank). A subtherapeutic course of CsA increased MST to 12.7+/-3 days (p<0.001 vs control). Combination with ABT-100 at 25 and 100 mg/kg BID improved MST to 18.7+/-5 days and 19.5+/-4 days (both p<0.001 vs control and respective monotherapy groups). ABT-100 treatment at 100 mg/kg BID significant decreased the amount of graft infiltrate (2.5+/-4 mononuclear cells/high power field (hpf) vs 29+/-11 cells/hpf, p<0.001). CONCLUSIONS: This is the first report that a specific FTI delays the development of acute rejection and supports the strategy of inhibiting Ras to impart immunomodulation. The antirejection and anticarcinogenic effects make FTIs a potentially useful adjunct in the antirejection regimens of malignancy-prone organ transplant recipients.

Development of a high-throughput robotic fluorescence-based assay for HsEg5 inhibitor screening.

HsEg5 has microtubule-activated ATPase activity and plays essential roles in bipolar spindle formation. Because HsEg5 is validated as an attractive cancer target, in vitro biochemical assays have been developed for identifying compounds with high inhibitory activity. Several compounds, including quinazoline ring-containing compounds, have been identified and are currently in clinical trials. Although considerable progress has been made during recent years, limitations of HsEg5 in vitro screening assays still reside in two main aspects. First, colorimetric-based assays exhibit relatively low sensitivity and limited dynamic range that are unable to accurately measure compounds with nanomolar potencies. Second, current fluorescence assays are relatively low throughput without "mix and read" homogeneous features. In this study, we describe a sensitive fluorescence-based assay for HsEg5-specific inhibitors. By coupling several enzymes' activities, the release of ADP was measured quantitatively through red fluorescent resorufin. The Km for ATP hydrolysis in this assay was calculated as 23 microM. The known HsEg5 inhibitors CK0106023 and CK0238273 gave IC50 values of 9.8 and 30.6 nM, respectively. Our fluorescence assay has a 20-fold increase in sensitivity with broader dynamic range when compared with a colorimetric assay. We further automated this assay for high-throughput screening with a Z' factor of 0.8.

Novel one-pot total syntheses of deoxyvasicinone, mackinazolinone, isaindigotone, and their derivatives promoted by microwave irradiation.

[reaction: see text]. Total syntheses of deoxyvasicinone (1), mackinazolinone (2), and 8-hydroxydeoxyvasicinone (3) via novel microwave-assisted domino reactions, as well as a novel three-component one-pot total synthesis of isaindigotone (5) promoted by microwave irradiation, are reported. The efficient reaction process enabled us to rapidly access related natural product derivatives and to identify a new class of cytotoxic agents.

An inhibitor of Bcl-2 family proteins induces regression of solid tumours.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

Discovery of novel inhibitors of Bcl-xL using multiple high-throughput screening platforms.

Bcl-xL is a member of the Bcl-2 family of proteins that are implicated to play a vital role in several diseases including cancer. Bcl-xL suppresses apoptosis; thus the inhibition of Bcl-xL function could restore the apoptotic process. To identify antagonists of Bcl-xL function, two ultra-high-throughput screens were implemented. An activity assay utilized fluorescence polarization, based on the binding of fluorescein-labeled peptide [the BH3 domain of BAD protein (F-Bad 6)] to Bcl-xL. A 384-well plate assay with mixtures of 10 drug compounds per well, combined with a fast plate reader, resulted in a throughput of 46,080 data points/day. Utilizing this screening format, 370,400 compounds were screened in duplicate and 425 inhibitors with an IC(50) below 100 microM were identified. The second assay format, affinity selection/mass spectrometry (ASMS), used ultrafiltration to separate Bcl-xL binders from nonbinders in mixtures of 2400 compounds. The bound species were subsequently separated from the protein and analyzed by flow injection electrospray mass spectrometry. Utilizing the ASMS format, 263,382 compounds were screened in duplicate and 29 binders with affinities below 100 microM were identified. Two novel classes of Bcl-xL inhibitors were identified by both methods and confirmed to bind (13)C-labeled Bcl-xL using heteronuclear magnetic resonance spectroscopy.

Divergence of Genbank and human tumor Bcl-2 sequences and implications for binding affinity to key apoptotic proteins.

Heterodimerization of antiapoptotic and pro-apoptotic Bcl-2 family of proteins provides an important mechanism for apoptosis regulation. Knowledge about key amino acids in the binding groove of native Bcl-2 contributing to this interaction will greatly facilitate the design of Bcl-2-specific inhibitors. There are two different Bcl-2 sequences, M13994 and M14745, in Genbank. Chimeric proteins Bcl-2(1) and Bcl-2(2) derived from the above sequences, although similar in structure, showed different binding affinities to Bak and Bad BH3 peptides (Petros et al., 2001). In this study, we show that the Bcl-2(1) sequence in normal and tumor human tissue samples differs from M13994 and M14745, and contains P59, T96, R110, S117 and G237. The actual sequence in the binding pocket matches the Bcl-2-Ig fusion sequence X06487, originally identified in a t(14:18) translocation of the Bcl-2 gene, associated with follicular lymphoma. The possible effects of the observed amino acid differences compared to M13994 and M14745 were investigated by combining structural data with fluorescence anisotropy. G110R substitution confers on Bcl-2(1) substantially increased binding affinity to Bak, Bad and Bax BH3 peptides, demonstrating that R110 is a key contributor to the BH3 binding affinity of Bcl-2. Although NMR structure did not predict R110 involvement in binding to these BH3 peptides, fluorescence anisotropy data clearly points to a critical role for this residue in binding to pro-apoptotic Bcl-2 family members.

Chkl binds and phosphorylates BAD protein.

Chk1 (checkpoint kinase 1) is a serine-threonine kinase that is critical for G2/M arrest in response to DNA damage. Chk1 phosphorylates Cdc25C at serine-216, a major regulatory site, in response to DNA damage. Furthermore, Chk1 also phosphorylates Cdc25A on serine 123 which accelerates its degradation through the ubiquitin-proteasome pathway and arrests cells in late G2-phase after DNA damage. In the present study, we demonstrated that Chk1 phosphorylates pro-apoptotic protein BAD (Bcl-2/Bcl-XL-Antagonist, causing cell Death) in vitro. In vitro phosphorylation analysis with various mouse BAD peptides has revealed two phosphorylation sites for Chk1 at serine-155 and serine-170. When wild-type and mutant BAD (S155A) constructs were transfected into 293T cells, an association between BAD and Chk1 was observed by co-immunoprecipitation. In addition, there was an increase in the phosphorylation of serine-155 following DNA damage by adriamycin treatment. Our results suggest that Chk1 associates with BAD and phosphorylates the BAD protein at serine-155. Taken together, our results suggest that Chk1 may inactivate BAD by associating with and phosphorylating residues critical for BAD function in response to DNA damage.