Antiproliferative and antitumor activities of PF-04691502 through the modulation of the PI3K/mTOR pathway.

<p><i>A</i>. Representative dose-dependent responses of CSCs and differentiated cells after treatment with PF-04691502. Cell viability, as measured by CellTiter Glo® assay, is shown as the mean ± SEM (n = 5). <i>B</i>. Tumor growth inhibition induced by the treatment with PF-04691502 in SCID-bg mice in comparison with the vehicle controls. *P≤0.05; **P≤0.001 (two-way ANOVA analysis). <i>C.</i> Relative change in body weight of mice treated in the experiment (%). <i>D.</i> Western blot analysis of treated xenograft tumors. The expression levels of pAKT (S473), pERK (T202/T204), and the loading control α-tubulin are shown in four individual xenograft tumors in each treatment group. <i>E.</i> Quantitative data of the expression levels of pAKT (S473) relative to the loading controls (mean ± SEM; n = 4). F. CSCs were treated with PF-04691502 (502) at 0.1, 0.5 and 1 µM in vitro for 3, 24, and 48 hours. The changes in pAKT (S473) and pERK (T202/T204) were evaluated by Western Blot. GAPDH served as a loading control.</p

Culture of spheroid CSCs and identification of CD133+/EpCAM+ cells.

<p><i>A</i>, Schema illustrating the work flow of spheroid CSC generation and differentiated cell populations as an experimental system, including the transplantation of a patient tumor into NOD/SCID mice, propagation of CSCs from P₁ xenograft tumors (spheroid; 10× objective magnification), and differentiation of CSCs into adherent cells with epithelial morphology (differentiated; 20×). <i>B</i>. Flow cytometric analysis of the primary cells isolated from the original patient tumor. PI staining excludes the dead cells (a). APC- and PE-conjugated isotype controls are shown in (b). A population of CD133+/EpCAM+ cells was detected (c). <i>C</i>. FACS of CD133+/EpCAM+ colon CSCs from the primary cell population derived from P₁ xenograft tumors. Dead cells and murine cells were first excluded by PI staining and using an anti-mouse specific monoclonal antibody H-2k[d], respectively (a & b). CD133+/EpCAM+ and CD133−/EpCAM+ populations were gated according to the baselines of isotype controls and sorted (c). Finally, enrichment of both populations in the sorted samples was confirmed by flow cytometry (d & e). <i>D</i>. Expression of the CSC markers in a fraction of cultured spheroid CSCs (right panel). Left panel shows isotype controls.</p

Enhanced tumorigenic potential of CSCs and histology of xenograft tumor tissues.

<p><i>A</i>. Tumor volumes in the <i>in vivo</i> limiting dilution assay for CSCs and their differentiated progeny in SCID-bg mice as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067258#s2" target="_blank">Materials and Methods</a> (mean ± SEM; n = 5). Differences in the growth rates were found between the CSC and differentiated cell groups inoculated with higher cell numbers (1×10⁶ and 1×10⁵; slope difference in lineage regression; P<0.01). <i>B</i>. Tumor take rates after 63 days post-implantation for CSCs and differentiated progeny. <i>C</i>. H&E stained sections of the primary patient tumor (<i>a</i>) and xenograft tumors derived from CSCs (<i>b</i>) and differentiated cells (<i>c</i>; upper panel, 40×; lower panel, 4×). Signet-ring cells were frequently identified in both patient and CSC-driven tumors and are indicated by arrows (a and b at 40×). A dot plot shows the frequency of signet-ring cells identified in the patient tumor, the CSC-derived xenograft and the differentiated cells-derived xenograft (d).</p

Self-renewal of CSCs and mutation detection.

<p><i>A</i>. Flow cytometric analysis of the CD133+/EpCAM+ fraction in xenograft tumors generated by CSCs and differentiated progeny. Data are shown as the CSC frequency representing the percentage of CD133+/EpCAM+ cells in tumors of two distinct origins (mean ± SEM, n = 3; unpaired, two-tailed student t-test, P<0.05). <i>B</i>. Re-implantation of tumor fragments obtained from CSCs or differentiated cell-derived xenograft tumors in secondary SCID-bg mice. Tumor volumes are shown as the mean ± SEM (n = 5). The difference in the growth curves between the two groups is statistically significant (difference in slope by linear regression; P<0.05). <i>C</i>. Cell proliferation of the primary cells isolated from either CSCs or differentiated cell-derived xenograft tumors in the stem-cell culture condition. Viable cells were plated at 20,000 cells per well and cultured for 17 days. Cell numbers were manually counted, and total cell counts are shown as the mean ± SEM (n = 6; unpaired, two-tailed student t-test, P<0.01). <i>D</i>. Spheroid CSC cultures re-established under the stem cell conditions from xenograft tumors generated by the serial implantation of CSC-derived xenograft tumors. P₂, CSCs cultured from xenograft tumors derived from the original CSCs. P₃, CSCs cultured from xenograft tumors derived from the P₂ CSCs. <i>E</i>. Partial MALDI-TOF mass spectrum showing the H1047R mutation in <i>PIK3CA</i>. The red dotted lines indicate, from left to right, the unextended primer peak, the 3 potential peaks for the mutant G or T alleles and the wild type A allele. The high peak in the middle of the figure is a T allele of <i>PDGFRA</i> in the same MALDI-TOF mass spectrum.</p

Enhanced tumorigenic potential of CD133+/EpCAM+ CSCs, CSC differentiation and drug resistance.

<p><i>A</i>. Average tumor volumes in NOD/SCID mice inoculated with CD133+/EpCAM+ (3,400 cell/animal; putative CSCs) or CD133−/EpCAM+ (25,000 cell/animal; putative differentiated) cells after 12 weeks of implantation (mean±SEM; n = 5). <i>B</i>. Percentage of CD133+/EpCAM+ expressing cells in the CSC and differentiated cell populations (mean ± SEM, n = 3; unpaired, two-tailed student t-test, P<0.05). <i>C</i>. Representative results of cell proliferation rates and expression levels of cytokeratin in CSCs and differentiated cells. Cell numbers on Y axes are adjusted as a percentage of the maximum cell numbers analyzed. <i>D</i>. Representative data showing the <i>in vitro</i> drug sensitivity of CSCs and their differentiated progeny in response to oxaliplatin as assessed by CellTiter Glo® assay.</p

Discovery of a Novel Class of Exquisitely Selective Mesenchymal-Epithelial Transition Factor (c-MET) Protein Kinase Inhibitors and Identification of the Clinical Candidate 2‑(4-(1-(Quinolin-6-ylmethyl)‑1<i>H</i>‑[1,2,3]triazolo[4,5‑<i>b</i>]pyrazin-6-yl)‑1<i>H</i>‑pyrazol-1-yl)ethanol (PF-04217903) for the Treatment of Cancer

The c-MET receptor tyrosine kinase is an attractive oncology target because of its critical role in human oncogenesis and tumor progression. An oxindole hydrazide hit <b>6</b> was identified during a c-MET HTS campaign and subsequently demonstrated to have an unusual degree of selectivity against a broad array of other kinases. The cocrystal structure of the related oxindole hydrazide c-MET inhibitor <b>10</b> with a nonphosphorylated c-MET kinase domain revealed a unique binding mode associated with the exquisite selectivity profile. The chemically labile oxindole hydrazide scaffold was replaced with a chemically and metabolically stable triazolopyrazine scaffold using structure based drug design. Medicinal chemistry lead optimization produced 2-(4-(1-(quinolin-6-ylmethyl)-1<i>H</i>-[1,2,3]­triazolo­[4,5-<i>b</i>]­pyrazin-6-yl)-1<i>H</i>-pyrazol-1-yl)­ethanol (<b>2</b>, <b>PF-04217903</b>), an extremely potent and exquisitely selective c-MET inhibitor. <b>2</b> demonstrated effective tumor growth inhibition in c-MET dependent tumor models with good oral PK properties and an acceptable safety profile in preclinical studies. <b>2</b> progressed to clinical evaluation in a Phase I oncology setting