Tracheoesophageal fistula closed by chemoradiotherapy in lung cancer.

A 45-year-old man complaining of cough, dyspnea, and difficulty in swallowing was referred to our hospital. Chest CT scan showed a mediastinal mass compressing the trachea. He was diagnosed with poorly differentiated lung carcinoma by percutaneous needle biopsy. Bronchoscopy and upper gastrointestinal endoscopy revealed a tracheoesophageal fistula (TEF). Long-lasting febrile neutropenia made it impossible to continue chemotherapy, but a course of radiotherapy (total 61 Gy) was completed. The next endoscopy revealed closure of the TEF. Chemoradiotherapy (CRT) has been reported to close TEF in esophageal cancer, but the risk of a CRT-induced worsening of the fistula has dissuaded physicians from using CRT to treat TEF in lung cancer patients. CRT may serve as a palliative treatment for TEF in lung cancer as well as esophageal cancer.

Potent anti-tumor activity of a macrocycle-quinoxalinone class pan-Cdk inhibitor in vitro and in vivo.

Deregulation of cell-cycle control is a hallmark of cancer. Thus, cyclin-dependent kinases (Cdks) are an attractive target for the development of anti-cancer drugs. Here, we report the biological characterization of a highly potent pan-Cdk inhibitor with a macrocycle-quinoxalinone structure. Compound M inhibited Cdk1, 2, 4, 5, 6, and 9 with equal potency in the nM range and was selective against kinases other than Cdks. This compound inhibited multiple events in the cell cycle in vitro, including retinoblastoma protein (pRb) phosphorylation, E2F-dependent transcription, DNA replication (determined by bromodeoxyuridine incorporation), and mitosis completion (assayed by flow cytometry) in the 10 nM range. Moreover, this compound induced cell death, as determined by induction of the subG1 fraction, activated caspase-3, and anexin V. In vivo, Compound M showed anti-tumor efficacy at a tolerated dose. In a nude rat xenograft tumor model, an 8-h constant infusion of Compound M inhibited pRb phosphorylation and induced apoptosis in tumor cells at ~ 30 nM, which led to the inhibition of tumor growth. Immunosuppression was the only liability observed at this dose, but immune function returned to normal after 10 days. Suppression of pRb phosphorylation in tumor cells was clearly correlated with tumor cell growth inhibition and cell death in vitro and in vivo. In vivo, Compound M inhibited pRb phosphorylation in both tumor and gut crypt cells. Rb phosphorylation may be a suitable pharmacodynamic biomarker in both tumors and normal tissues for monitoring target engagement and predicting the efficacy of Compound M.

Structures of the PKC-iota kinase domain in its ATP-bound and apo forms reveal defined structures of residues 533-551 in the C-terminal tail and their roles in ATP binding.

Protein kinase C (PKC) plays an essential role in a wide range of cellular functions. Although crystal structures of the PKC-theta, PKC-iota and PKC-betaII kinase domains have previously been determined in complexes with small-molecule inhibitors, no structure of a PKC-substrate complex has been determined. In the previously determined PKC-iota complex, residues 533-551 in the C-terminal tail were disordered. In the present study, crystal structures of the PKC-iota kinase domain in its ATP-bound and apo forms were determined at 2.1 and 2.0 A resolution, respectively. In the ATP complex, the electron density of all of the C-terminal tail residues was well defined. In the structure, the side chain of Phe543 protrudes into the ATP-binding pocket to make van der Waals interactions with the adenine moiety of ATP; this is also observed in other AGC kinase structures such as binary and ternary substrate complexes of PKA and AKT. In addition to this interaction, the newly defined residues around the turn motif make multiple hydrogen bonds to glycine-rich-loop residues. These interactions reduce the flexibility of the glycine-rich loop, which is organized for ATP binding, and the resulting structure promotes an ATP conformation that is suitable for the subsequent phosphoryl transfer. In the case of the apo form, the structure and interaction mode of the C-terminal tail of PKC-iota are essentially identical to those of the ATP complex. These results indicate that the protein structure is pre-organized before substrate binding to PKC-iota, which is different from the case of the prototypical AGC-branch kinase PKA.

Biological characterization of 2-aminothiazole-derived Cdk4/6 selective inhibitor in vitro and in vivo.

Abnormalities in the p16INK4a/ cyclin-dependent kinase (Cdk)4, 6/ Retinoblastoma (Rb) pathway frequently occur in various human cancers. Thus, Cdk4/6 is an attractive target for cancer therapy. Here we report the biological characterization of a 2-aminothiazole-derived Cdk4/6 selective inhibitor, named Compound A in vitro and in vivo. Compound A potently inhibits Cdk4 and Cdk6 with high selectivity (more than 57-fold) against other Cdks and 45 serine/threonine and tyrosine kinases. Compound A inhibits Rb protein (pRb) phosphorylation at Ser780, inhibits E2F-dependent transcription, and induces cell-cycle arrest at G1 in the T98G human glioma cell line. Among 82 human cells derived from various tissues, cell lines derived from hematological cancers (leukemia/lymphoma) tended to be more sensitive to Compound A in cell proliferation assay. Rb-negative cells tended to be insensitive to Compound A, as we had expected. In a nude rat xenograft model, Compound A inhibited pRb phosphorylation and bromodeoxyuridine (BrdU) incorporation in Eol-1 xenograft tumor at plasma concentration of 510 nM. Interestingly Compound A only moderately inhibited those pharmacodynamic and cell cycle parameters of normal crypt cells in small intestine even at 5 times higher plasma concentration. In F344 rats, Compound A did not cause immunosuppression even at 17 times higher plasma conc. These results suggest that Cdk4/6 selective inhibitors only moderately affects on the cell cycle of normal proliferating tissues and has a safer profile than pan-Cdk inhibitor in vivo.

Multiplexed random peptide library and phospho-specific antibodies facilitate human polo-like kinase 1 inhibitor screen.

One of the challenges to develop time-resolved fluorescence resonance energy transfer (TR-FRET) assay for serine/threonine (Ser/Thr) protein kinase is to select an optimal peptide substrate and a specific phosphor Ser/Thr antibody. This report describes a multiplexed random screen-based development of TR-FRET assay for ultra-high-throughput screening (uHTS) of small molecule inhibitors for a potent cancer drug target polo-like kinase 1 (Plk1). A screen of a diverse peptide library in a 384-well plate format identified several highly potent substrates that share the consensus motif for phosphorylation by Plk1. Their potencies were comparable to FKD peptide, a designed peptide substrate derived from well-described Plk1 substrate Cdc25C. A specific anti-phosphor Ser/Thr antibody p(S/T)F antibody that detects the phosphorylation of FKD peptide was screened out of 87 antibodies with time-resolved fluorometry technology in a 96-well plate format. Using FKD peptide and p(S/T)F antibody, we successfully developed a robust TR-FRET assay in 384-well plate format, and further miniaturized this assay to 1,536-well plate format to perform uHTS. We screened about 1.2 million compounds for Plk1 inhibitors using a Plk1 deletion mutant that only has the kinase domain and subsequently screened the same compound library using a full-length active-mutant Plk1. These uHTSs identified a number of hit compounds, and some of them had selectivity to either the deletion mutant or the full-length protein. Our results prove that a combination of random screen for substrate peptide and phospho-specific antibodies is very powerful strategy to develop TR-FRET assays for protein kinases.

Small-molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents.

Wee1 is a tyrosine kinase that phosphorylates and inactivates CDC2 and is involved in G(2) checkpoint signaling. Because p53 is a key regulator in the G(1) checkpoint, p53-deficient tumors rely only on the G(2) checkpoint after DNA damage. Hence, such tumors are selectively sensitized to DNA-damaging agents by Wee1 inhibition. Here, we report the discovery of a potent and selective small-molecule inhibitor of Wee1 kinase, MK-1775. This compound inhibits phosphorylation of CDC2 at Tyr15 (CDC2Y15), a direct substrate of Wee1 kinase in cells. MK-1775 abrogates G(2) DNA damage checkpoint, leading to apoptosis in combination with DNA-damaging chemotherapeutic agents such as gemcitabine, carboplatin, and cisplatin selectively in p53-deficient cells. In vivo, MK-1775 potentiates tumor growth inhibition by these agents, and cotreatment does not significantly increase toxicity. The enhancement of antitumor effect by MK-1775 was well correlated with inhibition of CDC2Y15 phosphorylation in tumor tissue and skin hair follicles. Our data indicate that Wee1 inhibition provides a new approach for treatment of multiple human malignancies.

Preferences for phosphorylation sites in the retinoblastoma protein of D-type cyclin-dependent kinases, Cdk4 and Cdk6, in vitro.

D-type cyclin-dependent kinases (Cdk4 and Cdk6) regulate the G1 to S phase progression of the mammalian cell cycle. It has been suggested that Cdk4 and Cdk6 may have distinct functions in vivo, even though they are indistinguishable biochemically. Here we show that although these Cdks phosphorylate multiple residues in pRB, they do so with different residue selectivities in vitro; Thr821 and Thr826 are preferentially phosphorylated by Cdk6 and Cdk4, respectively. This raises the possibility different substrate specificities lead to their different roles in the regulation of cellular events. Furthermore, our results indicate the new concept that Cdk itself contributes to substrate recognition.

Cdk-mediated phosphorylation of pRB regulates HDAC binding in vitro.

Retinoblastoma protein (pRB) controls the G1/S transition in the cell cycle by binding and inactivating E2F transcription factor. pRB changes the chromatin structure at the E2F-responsive promoter by recruiting histone deacetylase (HDAC) to the pRB-E2F complex, thus controlling the transcriptional activity of E2F. Cyclin-dependent kinases (Cdks) phosphorylate pRB and disrupt association between pRB and E2F. We investigated the effects of pRB phosphorylation on HDAC-1 binding in vitro. Phosphorylation of pRB by Cdk4-cyclin D2, Cdk2-cyclin E, and Cdk2-cyclin A inhibited association of pRB with HDAC. Among these Cdks, Cdk4-cyclin D2 showed particularly effective inhibition of pRB-HDAC complex formation. Using pRB mutants with various deletions in the N- and C-terminal domains, we found that both the pocket and C-terminal domains are important for regulating association between pRB and HDAC.