Positron emission tomography (PET) imaging with the amino acid tracer 6-18F-fluoro-l-3,4-dihydroxy-phenylalanine (18F-DOPA) may provide better spatial and functional information in human gliomas than CT or MRI alone. The l-type amino acid transporter 1 (LAT1) is responsible for membrane transport of large neutral amino acids in normal cells. This study assessed the relationship between LAT1 expression and 18F-DOPA uptake in human astrocytomas. Endogenous LAT1 expression was measured in established glioblastoma (GBM) cell lines and primary GBM xenografts using Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Uptake of 18F-DOPA was approximated in vitro using 3H-l-DOPA as an analog. Uptake of 3H-l-DOPA was assessed in cells expressing LAT1 shRNA or LAT1 siRNA and compared to non-targeted (NT) control shRNA or siRNA sequences, respectively. To demonstrate the clinical relevance of these findings, LAT1 immunofluorescence staining was compared with corresponding regions of 18F-DOPA PET uptake in patients with newly diagnosed astrocytomas. LAT1 mRNA and protein expression varies in GBM, and the extent of 3H-l-DOPA uptake was positively correlated with endogenous LAT1 expression. Stable shRNA-mediated LAT1 knockdown in T98 and GBM28 reduced 3H-l-DOPA uptake relative to NT shRNA by 57 (P < 0.0001) and 52 % (P < 0.001), respectively. Transient siRNA-mediated LAT1 knockdown in T98 reduced 3H-l-DOPA uptake relative to NT siRNA up to 68 % (P < 0.01). In clinical samples, LAT1 expression positively correlated with 18F-DOPA PET uptake (P = 0.04). Expression of LAT1 is strongly associated with 3H-l-DOPA uptake in vitro and 18F-DOPA uptake in patient biopsy samples. These results define LAT1 as a key determinant of 18F-DOPA accumulation in GBM.

Here we report that GNE-783, a novel checkpoint kinase-1 (CHK1) inhibitor, enhances the activity of gemcitabine by disabling the S-and G 2 cell-cycle checkpoints following DNA damage. Using a focused library of 51 DNA-damaging agents, we undertook a systematic screen using three different cell lines to determine which chemotherapeutics have their activity enhanced when combined with GNE-783. We found that GNE-783 was most effective at enhancing activity of antimetabolite-based DNA-damaging agents; however, there was a surprisingly wide range of activity within each class of agents. We, next, selected six different therapeutic agents and screened these in combination with GNE-783 across a panel of cell lines. This revealed a preference for enhanced chemopotentiation of select agents within tumor types, as, for instance, GNE-783 preferentially enhanced the activity of temozolomide only in melanoma cell lines. Additionally, although p53 mutant status was important for the overall response to combinations with some agents; our data indicate that this alone was insufficient to predict synergy. We finally compared the ability of a structurally related CHK1 inhibitor, GNE-900, to enhance the in vivo activity of gemcitabine, CPT-11, and temozolomide in xenograft models. GNE-900 significantly enhanced activity of only gemcitabine in vivo, suggesting that strong chemopotentiation in vitro can translate into chemopotentiation in vivo. In conclusion, our results show that selection of an appropriate agent to combine with a CHK1 inhibitor needs to be carefully evaluated in the context of the genetic background and tumor type in which it will be used. Mol Cancer Ther; 12(11); 2285-95. Ó2013 AACR.

Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G 2 -M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabine's antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor. Mol Cancer Ther; 12(10); 1968-80. Ó2013 AACR.

Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.

Purpose In breast cancer patients, Mailliez and others described that 5 of 70 patients (7%) developed a bevacizumab-induced nasal septal perforation. However, to date, no studies have reported such rates in colorectal cancer patients, who derive a survival advantage with this drug. Methods This study examined the incidence of bevacizumab-induced, clinically symptomatic, otolaryngology specialist-confirmed nasal septal perforation among 100 patients who had been consecutively-treated for metastatic colorectal cancer. Results The incidence of nasal septal perforation was 1% (95% confidence intervals: −0.95% to 2.95%). This single adverse event was successfully managed conservatively. Within the whole group, 94 had been treated with bevacizumab at 5 mg/kg every two weeks, except for four patients treated at higher doses. The median number of bevacizumab doses (range) was seven (1–96). Concomitant chemotherapy had been prescribed to all patients, consisting of oxaliplatin, 5-fluorouracil, leucovorin, as per one of the FOLFOX regimens (44 patients); irinotecan, 5-fluorouracil, leucovorin, as per the FOLFIRI regimen (13 patients); both these regimens and no other (five patients); or a different regimen (38 patients). Conclusion Nasal septal perforation from bevacizumab occurs infrequently among colorectal cancer patients.