Motonobu Osada

Screening the p53 status of human cell lines using a yeast functional assay

We have screened the p53 status of 156 human cell lines, including 142 tumor cell lines from 27 different tumor types and 14 cell lines from normal tissues by using functional analysis of separated alleles in yeast. This assay enables us to score wild‐type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS3 via the p53‐responsive GAL1 promoter in Saccharomyces cerevisiae. Of 142 tumor cell lines, at least 104 lines (73.2%) were found to express the mutated p53 gene: 94 lines (66.2%) were mutated in both alleles, three lines (2.1%) were heterozygous, and no p53 cDNA was amplified from seven lines (4.9%). Of the 14 cell lines originating from normal tissues, all the transformed or immortalized cell lines expressed mutant p53 only. Yeast cells expressing mutant p53 derived from 94 cell lines were analyzed for temperature‐sensitive growth. p53 cDNA from eight cell lines showed p53‐dependent temperature‐sensitive growth, growing at 30°C but not at 37°C. Four temperature‐sensitive p53 mutations were isolated: CAT→CGT at codon 214 (H214R), TAC→TGC at codon 234 (Y234C), GTG→ATG at codon 272 (V272M), and GAG→AAG (E285K). Functionally wild‐type p53 was detected in 38 tumor cell lines (26.8%) and all of the diploid fibroblasts at early and late population doubling levels. These results strongly support the previous findings that p53 inactivation is one of the most frequent genetic events that occurs during carcinogenesis and immortalization. Mol. Carcinog. 19:243–253, 1997.

THE AH RECEPTOR IS NOT INVOLVED IN 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN-MEDIATED APOPTOSIS IN HUMAN LEUKEMIC T CELL LINES

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental pollutant causing public concern. Its toxic effects include disruption of the immune, endocrine, and reproductive systems, impairment of fetal development, carcinogenicity, and lethality in rodents. Here, we report that TCDD induces apoptosis in two cultured human leukemic lymphoblastic T cell lines. This cell death was found not to be dependent on an aryl hydrocarbon receptor and to be inhibited by the inhibitor of tyrosine kinases and caspases. Apoptosis-linked c-Jun N-terminal kinase is rapidly activated in these cells by the treatment with TCDD. A dominant-negative mutant of c-Jun N-terminal kinase prevented cell death in the treatment with TCDD. Furthermore, TCDD decreases the Bcl-2 protein level in these cell lines. These findings will help in the understanding of the molecular mechanism underlying TCDD-mediated immunotoxicity.

Assessment of cancer susceptibility in humans by use of genetic polymorphisms in carcinogen metabolism.

Prevention is an important and effective measure for reducing death caused by cancer. Thus information on individual susceptibility to cancer is valuable in suggesting high risk individuals to avoid intake of carcinogenic substances and receive frequent physical screening. To this end, polymorphisms found within cytochrome P450 (CYP) genes implicated in the metabolism of procarcinogens are expected to be good genetic targets in assessing human cancer susceptibility. We have found polymorphisms in the CYP2E1 and CYP1A1 genes associated with lung cancer susceptibility, though there were some discrepancies from observations made by other investigators. Discrepancies among investigators from different regions, however, are very common in these pharmacogenetic studies. We present an explanation for these discrepancies, difficulties associated with prediction of relative risk of individuals, and future directions.

Abstract CT094: Phase I study of fibroblast growth factor receptor 2 antibody-drug conjugate (FGFR2-ADC) BAY 1187982 in patients with advanced cancer

Background: FGFR2-ADC (BAY 1187982) consists of an anti-FGFR2 antibody (BAY 1179470) and a stably linked, highly potent and non-cell permeable microtubule-binding auristatin toxophore. A phase I study was conducted in patients (pts) with advanced solid tumors focusing on tumor types with a described expression of FGFR2. Methods: Eligible pts with solid tumors, known to express FGFR2 and refractory to standard treatment, adequate organ function, and ECOG performance status (PS) ≤ 1 were enrolled. BAY 1187982 was administered by an one hour intravenous infusion on an every 21 days (q3w) schedule in 20 pts (5 cholangiocarcinoma, 5 colon, 2 gastric, 2 breast cancer and 6 others) at doses ranging from 0.1 to 1.3 mg/kg. Dose escalation followed the continous reassessment design treating 3-4 pts per cohort. Pts were assessed for AEs weekly in C1-C3 and on D1 in subsequent cycles. Tumor response was assessed every 6 weeks. Blood samples for a full pharmacokinetic profile were collected after dosing on D1 of C1 and C3 and thereafter every second cycle pre-dose and at the end of infusion. Results: A total of 20 pts were evaluable: 55% female, mean age 52 yrs (range 24-76), PS 0/1 75% / 25%. No drug-related serious adverse events were reported at dose levels 0.1 and 0.2 mg/kg. At 0.4 mg/kg drug-related serious adverse events were similar to other ADCs, including reversible asymptomatic liver function test increases, and gastrointestinal disorders. Dose limiting toxicities (DLTs) were observed at dose level 0.8 and 1.3 mg/kg, respectively: 0.8 mg/kg cohort grade 3 thrombocytopenia (N=1); 1.3 mg/kg cohort grade 3 corneal epitheliopathy (N=1), grade 4 thrombocytopenia (N=1) and proteinuria progressing to nephrotic syndrome after treatment in C2 or C3 (N=2) with signs of reversibility after discontinuation. In dose level 0.4 and 0.8 mg/kg the majority of pts discontinued study drug treatment already in C1 or C2 due to disease progression and data on proteinuria at these dose levels were absent or spare. As a consequence only doses below 0.4 mg/kg were considered safe. Exposures of BAY 1187982 increased in a dose-proportional manner in the investigated dose range from 0.1 to 1.3 mg/kg. Conclusion: While this drug was tolerated well at lower dose levels, dose-limiting toxicities occurred below the expected therapeutic dose level. As the predicted human efficacious dose level based on the majority of animal models was above 0.4 mg/kg (Wittemer-Rump et al. AACR; Cancer Res 2015;75, 15 Suppl: Abstract nr 1683) a Phase II dose cannot be recommended and a successful clinical development of the compound was regarded as unlikely. The data from the preclinical safety analysis in monkey and rat will be submitted as abstract to AACR 2017. Citation Format: Sung-Bae Kim, Funda Meric-Bernstam, Jordan Berlin, Sabine Wittemer-Rump, Motonobu Osada, Ray Valencia, Aleksei Babich, Rong Liu, Andy Hwang, Takahiko Tanigawa, Frank Reetz, Dirk Laurent, Aparna Kalyan. Phase I study of fibroblast growth factor receptor 2 antibody-drug conjugate (FGFR2-ADC) BAY 1187982 in patients with advanced cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT094. doi:10.1158/1538-7445.AM2017-CT094