Claudia Wilm

Quantitative live imaging of cancer and normal cells treated with Kinesin-5 inhibitors indicates significant differences in phenotypic responses and cell fate

Kinesin-5 inhibitors (K5I) are promising antimitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using time-lapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and noncancer cell types to undergo prolonged arrest in monopolar mitosis. Subsequent events, however, differed greatly between cell types. Normal diploid cells mostly slipped from mitosis and arrested in tetraploid G1, with little cell death. Several cancer cell lines died either during mitotic arrest or following slippage. Contrary to prevailing views, mitotic slippage was not required for death, and the duration of mitotic arrest correlated poorly with the probability of death in most cell lines. We also assayed drug reversibility and long-term responses after transient drug exposure in MCF7 breast cancer cells. Although many cells divided after drug washout during mitosis, this treatment resulted in lower survival compared with washout after spontaneous slippage likely due to chromosome segregation errors in the cells that divided. Our analysis shows that K5Is cause cancer-selective cell killing, provides important kinetic information for understanding clinical responses, and elucidates mechanisms of drug sensitivity versus resistance at the level of phenotype. [Mol Cancer Ther 2008;7(11):3480–9]

Matched rabbit monoclonal antibodies against αv-series integrins reveal a novel αvβ3-LIBS epitope, and permit routine staining of archival paraffin samples of human tumors.

Summary The relationship between integrin expression and function in pathologies is often contentious as comparisons between human pathological expression and expression in cell lines is difficult. In addition, the expression of even integrins &agr;v&bgr;6 and &agr;v&bgr;8 in tumor cell lines is not comprehensively documented. Here, we describe rabbit monoclonal antibodies (RabMabs) against the extracellular domains of &agr;v integrins that react with both native integrins and formalin fixed, paraffin embedded (FFPE) human tissues. These RabMabs, against &agr;v&bgr;3 (EM22703), &agr;v&bgr;5 (EM09902), &agr;v&bgr;6 (EM05201), &agr;v&bgr;8 (EM13309), and pan-&agr;v (EM01309), recognize individual integrin chains in Western blots and in flow cytometry. EM22703 detected a ligand-induced binding site (LIBS), reporting an epitope enhanced by the binding of an RGD-peptide to &agr;v&bgr;3. &agr;v&bgr;8 was rarely expressed in human tumor specimens, and weakly expressed in non-small-cell lung carcinoma (NSCLC). However, ovarian carcinoma cell lines expressed &agr;v&bgr;8, as did some melanoma cells, whereas U87MG glioma lacked &agr;v&bgr;8 expression. We observed an unexpected strong expression of &agr;v&bgr;6 in tumor samples of invasive ductal breast adenoma, colorectal carcinoma (CRC), and NSCLC. &agr;v&bgr;3 was strongly expressed in some invasive NSCLC cohorts. Interestingly, PC3 prostate cell and human prostate tumors did not express &agr;v&bgr;3. The RabMabs stained plasma membranes in FFPE-immunohistochemistry (IHC) samples of tumor cell lines from lung, ovary, colon, prostate, squamous cell carcinoma of head and neck (SCCHN), breast, and pancreas carcinomas. The RabMabs are unique tools for probing &agr;v integrin biology, and suggest that especially &agr;v&bgr;6 and &agr;v&bgr;8 biologies still have much to reveal.

2. Endothelin antagonists : Evaluation of 2,1,3-benzothiadiazole as a methylendioxyphenyl bioisoster

The methylendioxyphenyl group is present in a number of endothelin receptor antagonists thus far reported. By means of a Kohonen neural network we discovered with a benzothiadiazole a bioisosteric replacement instead. This group should be devoid of the negative metabolic interactions with cytochrome P450 ascribed to methylendioxyphenyl in vivo. The synthesis of a potent benzothiadiazole analogue EMD 122801 together with in vitro studies of different methylendioxyphenyl, benzothiadiazole and benzofurazan derivatives is described.

1,4-Diaryl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acids as endothelin receptor antagonists

Abstract The discovery, synthesis and structure-activity relationships of a series of novel 1,4-diaryl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acids as non-selective endothelin ETA / ETB receptor antagonists are described. The most potent inhibitor 3m displayed an IC50 of 260 nM and 200 nM for ETA and ETB receptors, respectively.

Benzofuro[3,2-b]pyridines as mixed ETAETB and selective ETB endothelin receptor antagonists

The discovery, synthesis and structure-activity relationships of a series of novel benzofuro[3,2-b]pyridines as non-selective endothelin ET(A)/ET(B) as well as selective ET(B) receptor antagonists are described. The most potent non-selective inhibitor 7s displayed an IC50 of 21 nM and 41 nM for ET(A) and ET(B) receptors, respectively, whereas 7ee merely showed affinity for the ET(B) receptor (IC50 = 3.6 nM).

3. Endothelin antagonists: discovery of EMD 122946, a highly potent and orally active ETA selective antagonist

The discovery, in vitro and in vivo studies of the highly potent ETA antagonist EMD 122946 are presented. This compound displayed high binding affinity and functional antagonism [IC50 = 3.2 x 10(-11) M, pA2 = 9.5 (ETA)] and inhibited the ET-1 induced pressor response in pithed rats with an ED50 of 0.3 mg/kg. In conscious spontaneously hypertensive rats and in DOCA-salt hypertensive rats the compound lowered mean blood pressure with an ED50 of 0.06 mg/kg. EMD 122946 exhibited high bioavailability in rats and monkeys.

Pyridazinones with a pendant acylsulfonamide moiety as endothelin receptor antagonists

Abstract Highly active endothelin receptor antagonists can be obtained by replacing the aryloxy group of L-749,329 by diversely substituted pyridazinone residues. The syntheses and structure-activity relationships of the new aryl-oxopyridazinyl-N-(4-arylsulfonyl)-acetamides 2 are reported. 2p with a simple dimethyl-pyridazinone moiety was one of the most potent compounds in vitro.

PD-L1 immunostaining scoring for non-small cell lung cancer based on immunosurveillance parameters

Non-Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death globally, and new immunotherapies developed and under development targeting PD-1/PD-L1 checkpoint inhibition require accurate patient selection to assure good clinical outcome. PD-L1 immunohistochemistry is the current biomarker assay used for patient selection, but still imprecise in predicting therapy response. Exploring this issue, we performed computational tissue analysis of PD-L1 immunostaining in procured NSCLC tissues (n = 50) using the Merck KGaA anti-PD-L1 clone MKP1A07310. Staining patterns and PD-L1 cut-off points were interrogated using relevant cancer immune-surveillance biomarkers. Groups with high PD-L1 expression levels (above 25/50% staining cut-off points) were enriched for a biomarker profile in the tumor-nest and microenvironment indicating escape from host-immunity, as represented by increased numbers of cells positive for CD8 and Granzyme B (immune-effectors), FOXP3 (immune-suppressive), and CD68 (P < 0.05). Manual analysis of PD-L1 staining patterns identified tumors with an immune-induced reactive pattern relevant for immunotherapy that would ordinarily be excluded by the arbitrary 25% staining threshold (P < 0.05). Conversely, some cases with completely or predominantly immune-independent constitutive PD-L1 staining patterns that indicate insensitivity to immunotherapy may have been incorrectly selected using this staining cut-off point criterion. Therefore, we propose differentiation of reactive vs constitutive PD-L1 staining patterns to improve the accuracy of this biomarker assay in selecting NSCLC patients for PD-1/PD-L1 immunotherapy.