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XLSX file - 32K, Phosphorylation of downstream intracellular kinases.

Although most children with acute myeloid leukemia (AML) achieve complete remission, the relapse rate is 30% to 40%. Because it is thought that leukemia-initiating cells (LIC) are responsible for AML relapses, targeting these cells might improve outcome. Treatment of pediatric AML blasts with the receptor tyrosine kinase (RTK) inhibitor PTK787/ZK 222584 (PTK/ZK) induces cell death in vitro. However, the role of PTK/ZK inhibition on outgrowth of (pediatric) LICs is unknown. In this study, we cultured CD34+ cells from pediatric patients with AML on MS5 stromal cells in long-term cocultures. In analogy to adult AML, long-term expansion of leukemic cells up to 10 weeks could be generated in 9 of 13 pediatric AMLs. Addition of PTK/ZK to long-term cocultures significantly inhibited leukemic expansion in all samples, ranging from 4% to 80% growth inhibition at week 5 compared with untreated samples. In 75% of the samples, the inhibitory effect was more pronounced at week 10. Proteome profiler array analysis of downstream kinases revealed that PTK/ZK reduced activation of PI3K/Akt kinase signaling. Although main targets of PTK/ZK are VEGF receptors (VEGFR), no effect was seen on outgrowth of LICs when cultured with bevacizumab (monoclonal VEGFA-antibody), specific antibodies against VEGFR2 or VEGFR3, or exposed to stroma-derived VEGFA. These data suggest that the effect of PTK/ZK on LICs is not only dependent on inhibition of VEGFA/VEGFR signaling. Taken together, our data elucidated antileukemic properties of PTK/ZK in long-term expansion cultures, and suggest that targeting multiple RTKs by PTK/ZK might be a potential effective approach in eradicating (pediatric) LICs. Mol Cancer Res; 11(4); 339–48. ©2013 AACR.

XLSX file - 21K, Specific primers for RT-PCR.

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PDF file - 147K, Effect of VEGFA-addition, Bevacizumab, specific VEGFR-inhibitor and MK2206 on CD34+ sorted pediatric AML cells.

PDF file - 161K, Effect of PTK787/ZK 222584 and MK2206 on AML cell lines.

Progression in pediatric brain tumor growth is thought to be the net result of signaling through various protein kinase-mediated networks driving cell proliferation. Defining new targets for treatment of human malignancies, without a priori knowledge on aberrant cell signaling activity, remains exceedingly complicated. Here, we introduce kinome profiling using flow-through peptide microarrays as a new concept for target discovery. Comprehensive tyrosine kinase activity profiles were identified in 29 pediatric brain tumors using the PamChip kinome profiling system. Previously reported activity of epidermal growth factor receptor, c-Met, and vascular endothelial growth factor receptor in pediatric brain tumors could be appreciated in our array results. Peptides corresponding with phosphorylation consensus sequences for Src family kinases showed remarkably high levels of phosphorylation compared with normal tissue types. Src activity was confirmed applying Phos-Tag SDS-PAGE. Furthermore, the Src family kinase inhibitors PP1 and dasatinib induced substantial tumor cell death in nine pediatric brain tumor cell lines but not in control cell lines. Thus, this study describes a new high-throughput technique to generate clinically relevant tyrosine kinase activity profiles as has been shown here for pediatric brain tumors. In the era of a rapidly increasing number of small-molecule inhibitors, this approach will enable us to rapidly identify new potential targets in a broad range of human malignancies. [Cancer Res 2009;69(14):5987–95]

Supplementary Table 3 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Figure Legend from Repression of Vascular Endothelial Growth Factor Expression by the Runt-Related Transcription Factor 1 in Acute Myeloid Leukemia

Supplementary Figure 3 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Figure 2 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Table 4 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Figure 1 from Repression of Vascular Endothelial Growth Factor Expression by the Runt-Related Transcription Factor 1 in Acute Myeloid Leukemia

Supplementary Figure 1 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Table 2 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Figure 4 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Supplementary Table 1 from Kinome Profiling in Pediatric Brain Tumors as a New Approach for Target Discovery

Background: In this national multicentre study, we examined the safety of reducing antibiotics in selected paediatric cancer patients with febrile neutropenia.Methods: Patients with signs of a bacterial infection and/or abnormal vital signs indicating sepsis were considered high risk and received antibiotic therapy. Remaining patients were allocated to low-or medium risk, depending on their interleukin-8 level. Low-risk patients did not receive any antibiotics and were discharged from the hospital after having been afebrile for 12 h. Medium-risk patients were re-evaluated after 72 h of antibiotic treatment and, in selected patients, antibiotics were stopped.Results: Two hundred thirty-three febrile neutropenic episodes in 141 paediatric cancer patients were included in the study. Sixty-four episodes were classified high risk (28%), 122 medium risk (52%), and 47 (20%) low risk. In the medium-risk group, antibiotics were stopped after 72 h in 50 in 122 episodes (41%). Median duration of antibiotic treatment and hospital admission was significantly lower in low-and medium-risk episodes with early discharge. No failures were observed in the medium-risk group with early discharge. In the low-risk group, six failures were observed (12.8%), due to coagulase-negative staphylococci-positive blood cultures and recurrent fever.Conclusion: We showed that it is safe to shorten antibiotic treatment to 72 h in selected medium-risk patients with febrile neutropenia, regardless of the neutrophil count. The safety of withholding antibiotics in selected low-risk paediatric cancer patients with febrile neutropenia requires further investigation, using more suitable definitions for safety. Reduction in hospital admissions allows children with cancer more time at home and consequently improves their quality of life. (C) 2015 Elsevier Ltd. All rights reserved.